MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'BJ Signal' source. Mon, 06 Feb 2012 13:31:36 +0100 http://www.medworm.com/index.php?rid=5664018&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20120035 The paracaspase domain of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of a gene translocation fused to the N-terminal domains of the cellular inhibitor of apoptosis protein 2. The paracaspase itself, commonly known as MALT1, participates in the NF-κB pathway, likely by driving survival signals downstream of the B-cell antigen receptor through MALT1 proteolytic activity. We have developed methods for the expression and purification of recombinant full-length MALT1 and its constituent catalytic domain alone. Both are activated by dimerization without cleavage, with a similar dimerization barrier to the distantly related cousins, the apical caspases. By using positional scanning peptidyl substrate libraries we demonstrate that the activity ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5664018 Mon, 06 Feb 2012 05:00:00 +0100 5664018 http://www.medworm.com/index.php?rid=5656184&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111644 The vital signalling molecule nitric oxide is produced by mammalian NOS enzymes in two steps. L-arginine is converted to N-hydroxy-L-arginine NOHA, which is converted to NO and citrulline. Both steps are thought to proceed via similar mechanisms in which the cofactor tetrahydrobiopterin (H4B) activates dioxygen at the heme site by electron transfer. The subsequent events are poorly understood due to the lack of stable intermediates. By analogy with cytochromes P450, a heme-iron oxo species may be formed, or direct reaction between a heme-peroxy intermediate and substrate may occur. The two steps may also occur via different mechanisms. Here we analyse the two reaction steps using the G586S mutant of nNOS, which introduces an additional H-bond in the active site and provides an additional p... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5656184 Fri, 03 Feb 2012 05:00:00 +0100 5656184 http://www.medworm.com/index.php?rid=5592692&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20112026 Recombinant muscle glycogen synthase-1 (GYS1) and recombinant liver glycogen synthase-2 (GYS2) were phosphorylated by recombinant AMP-activated protein kinase (AMPK) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favorable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (assayed in the absence of Glc-6-P) and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with pharmacological AMPK activators 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICA riboside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by gluca... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5592692 Wed, 11 Jan 2012 05:00:00 +0100 5592692 http://www.medworm.com/index.php?rid=5576490&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20112000 The canonical pathway of regulation of the germinal centre kinase (GCK) III subgroup member, mammalian Sterile20-related kinase 3 (MST3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr178-), induction of Ser-/Thr-protein kinase activity and nuclear localisation. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein, GOLGA2/gm130. Activation of MST3 by calyculin A (a protein Ser-/Thr- phosphatase 1/2A inhibitor) stimulated (auto)phos... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5576490 Mon, 09 Jan 2012 05:00:00 +0100 5576490 http://www.medworm.com/index.php?rid=5568546&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110921 The ErbB4 receptor tyrosine kinase possesses both tumor suppressor and oncogenic activities. Thus, pharmacologic agents are needed to help elucidate ErbB4 functions. However, limitations of existing ErbB4 agonists and antagonists have led us to seek novel ErbB4 antagonists. The Q43L mutant of the ErbB4 agonist NRG2beta stimulates ErbB4 tyrosine phosphorylation, yet fails to stimulate ErbB4 coupling to cell proliferation. Thus, here we hypothesize that NRG2beta/Q43L may be an ErbB4 antagonist. NRG2beta/Q43L competitively antagonizes agonist stimulation of ErbB4 coupling to cell proliferation. NRG2beta/Q43L stimulates less ErbB4 tyrosine phosphorylation than does NRG2beta. In addition, NRG2beta stimulation of cell proliferation requires PI3K activity and NRG2beta stimulates greater Akt ph... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5568546 Thu, 05 Jan 2012 05:00:00 +0100 5568546 http://www.medworm.com/index.php?rid=5568548&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111035 The autoinhibition/activation of the plasma membrane calcium pump (PMCA) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae, was investigated using the phosphatidylcholine analogue [125I]TID-PC/16, which was incorporated into mixtures of dimyristoyl-phosphatidylcholine and the nonionic detergent C12E10. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+-calmodulin of PMCA2 showed 30 times higher affinity than PMCA4. PMCA2 exhibited a lower level labeling i... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5568548 Wed, 04 Jan 2012 05:00:00 +0100 5568548 http://www.medworm.com/index.php?rid=5568547&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111530 Eukaryotic elongation factor 2 kinase (eEF2K) phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily but instead belongs to a small group of so-called a-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. Here we identified several autophosphorylation sites including Thr-348, Thr-353, Ser-366 and Ser-445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser-78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5568547 Wed, 04 Jan 2012 05:00:00 +0100 5568547 http://www.medworm.com/index.php?rid=5568549&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111280 Glucose metabolism in the liver activates transcription of various genes encoding enzymes of glycolysis and lipogenesis and also glucose 6-phosphatase (G6pc). Allosteric mechanisms involving glucose 6-P or xylulose 5-P and covalent modification of ChREBP have been implicated in this mechanism. However evidence supporting an essential role for a specific metabolite or pathway in hepatocytes remains equivocal. By using diverse substrates and inhibitors and a kinase-deficient bisphosphatase-active variant of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FBP2), we demonstrate an essential role for fructose 2,6-bisphosphate in the induction of G6pc and other ChREBP target genes by glucose. Selective depletion of fructose 2,6-bisphosphate inhibits glucose-in... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5568549 Tue, 03 Jan 2012 05:00:00 +0100 5568549 http://www.medworm.com/index.php?rid=5532957&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111867 The low density lipoprotein receptor-related protein (LRP) can bind a wide range of structurally diverse ligands to regions composed of clusters of ~40 residue Ca2+-dependent, disulfide-rich, complement-like repeats (CR). While lysine residues from the ligands have been implicated in binding, there has been no quantitation of the energetic contributions of such interactions and hence of their relative importance in overall affinity, or of the ability of arginine or histidine to bind. We have used four representative CR domains from the principal ligand cluster of LRP to determine the energetics of interaction with well defined small ligands, that include methyl esters of lysine, arginine, histidine, and aspartic acid, as well as N-terminally blocked lysine methyl ester. We found tha... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5532957 Tue, 20 Dec 2011 05:00:00 +0100 5532957 http://www.medworm.com/index.php?rid=5512097&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20112037 Herein, we have examined whether IκB Kinase β (IKKβ) plays a role in feedback inhibition of the insulin-signaling cascade. Insulin induces the phosphorylation of IKKβ, in vitro and in vivo, and this effect is dependent on intact signaling via phosphatidylinositol 3-kinase (PI3K), but not protein kinase B (PKB). To test the hypothesis that insulin activates IKKβ as a means of negative feedback, we employed a variety of experimental approaches. Firstly, pharmacological inhibition of IKKβ via BMS-345541 did not potentiate insulin-induced IRS1 tyrosine phosphorylation, PKB phosphorylation or 2-deoxyglucose uptake in differentiated 3T3-L1 adipocytes. BMS-345541 did not prevent insulin-induced IRS1 serine phosphorylation on known IKKβ target sites... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5512097 Wed, 14 Dec 2011 05:00:00 +0100 5512097 http://www.medworm.com/index.php?rid=5512096&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111647 Suppressor Of Cytokine Signalling (SOCS) proteins are negative feedback regulators of the Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT) pathway. Their expression levels are low in physiological conditions, but they are up-regulated in response to cytokine stimulation in many immune and inflammatory processes. Overexpression of SOCS1 in keratinocyte clones abrogates the IFN-γ-induced expression of many pro-inflammatory genes and the release of related chemokines by blocking the JAK-STAT pathway. SOCS1 inhibits JAK2 kinase activity by binding the catalytic site of JAK2, with its Kinase Inhibitory Region (KIR) acting as a pseudo-substrate of the enzyme. Here we screened a focused combinatorial peptide library of KIR to identify new peptides able to mim... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5512096 Wed, 14 Dec 2011 05:00:00 +0100 5512096 http://www.medworm.com/index.php?rid=5482762&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111341 Chemokine CXCL12 signaling through receptors CXCR4 and CXCR7 has essential functions in development and underlies diseases including cancer, atherosclerosis, and autoimmunity. Chemokines may form homodimers that regulate receptor binding and signaling, but previous studies with synthetic CXCL12 have produced conflicting evidence for homodimerization. We used bioluminescence imaging with Gaussia luciferase fusions to investigate dimerization of CXCL12 secreted from mammalian cells. By column chromatography and Gaussia luciferase complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signaling through Gαi and AKT, while ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5482762 Tue, 06 Dec 2011 05:00:00 +0100 5482762 http://www.medworm.com/index.php?rid=5482763&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111913 In in vitro studies class-I PI 3-kinases, class-II PI 3-kinases and mTOR have all been described as having roles in regulation of glucose metabolism. The relative role each plays in the normal signalling processes regulating glucose metabolism in vivo is less clear. Knockout and knockin mouse models have provided some evidence that class-I PI 3-kinase isoforms p110a, p110b, and to a lesser extent p110g, are necessary for processes regulating glucose metabolism and appetite. However, in these models the PI 3-kinase activity is chronically reduced. Therefore we analysed the effects of acutely inhibiting PI 3-kinase isoforms alone, or PI 3-kinase and mTOR, on glucose metabolism and food intake. In these studies impairments in glucose tolerance and insulin tolerance and increased hepatic gluco... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5482763 Mon, 05 Dec 2011 05:00:00 +0100 5482763 http://www.medworm.com/index.php?rid=5464995&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111466 The Saccharomyces cerevisiae Hal3 protein is a moonlighting protein, able to function both as an inhibitory subunit of the Ppz1 protein phosphatase and as a constituent protomer of an unprecedented heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC), the third enzyme of the CoA biosynthetic pathway. We initiated here the dissection of the structural elements required for both disparate cellular tasks by using a combination of biochemical and genetic approaches. We show that the conserved Hal3 core (the PPCDC domain) is necessary for both functions, as determined by in vitro and in vivo assays. The Hal3 N-terminal domain is not functional by itself, although in vitro experiments indicate that when this domain is combined with the core it has a relevant function in Hal3’s... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5464995 Tue, 29 Nov 2011 05:00:00 +0100 5464995 http://www.medworm.com/index.php?rid=5447459&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111418 We report that exposure of T47D cells to synthetic progestins (ORG2058 or Norgestrel) leads to a rapid increase in Fru-2,6-P2 concentration. Western blot results are compatible with a short-term activation due to PFKFB3 isoenzyme phosphorylation and a long-term sustained action due to increased PFKFB3 protein levels. Transient transfection of T47D cells with deleted gene promoter constructs allowed us to identify a progesterone regulatory element (PRE) to which progesterone receptor binds and thus transactivates PFKFB3 gene transcription. Progesterone Receptor (PR) expression, in the PR-negative cell line (MDA-MB-231), induces endogen PFKFB3 expression in response to Norgestrel. Direct Binding of PR to the PRE box (-3490 nt) was confirmed by ChIP experiments. A dual mechanism affecting PFK... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5447459 Thu, 24 Nov 2011 05:00:00 +0100 5447459 http://www.medworm.com/index.php?rid=5396489&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111005 Porphyrias are diseases caused by partial deficiencies of haem biosynthesis enzymes. Acute porphyrias are characterized by a neuropsychiatric syndrome with intermittent induction of hepatic δ-aminolevulinate synthase 1 (ALAS-1), first and rate-limiting enzyme of haem pathway. Porphyria acute attacks are usually treated with glucose administration, its effect is apparently related to its ability of inhibiting ALAS-1 by modulating insulin plasma levels. It was shown that insulin blunts hepatocytes ALAS-1 induction, by disrupting the interaction of the Forkhead box O1 (FOXO1) and the proliferator-activated receptor γ coactivator 1α (PGC-1α). We evaluated the expression of ALAS-1 in a murine model of diabetes and determined the effects of the insulinomimetic vanadat... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5396489 Thu, 10 Nov 2011 05:00:00 +0100 5396489 http://www.medworm.com/index.php?rid=5396490&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111664 G-protein-regulated phosphoinositide 3-kinase g (PI3Kg) plays a crucial role in inflammatory and allergic processes. PI3Kγ, a dimeric protein formed by non-catalytic p101 and catalytic p110γ subunits, is stimulated by receptor-released Gbg complexes. We previously demonstrated that Gbg stimulates both monomeric p110g and dimeric p110g/p101 lipid kinase activity in vitro. In order to identify the Gb residues responsible for the Gbg-PI3Kg-interaction, we examined Gb1 mutants for their ability to stimulate lipid and protein kinase activities and to recruit PI3Kg to lipid vesicles. Our findings revealed different interaction profiles of Gβ residues interacting with p110g or p110g/p101. Moreover, p101 was able to rescue the stimulatory activity of Gb1 mutants incapable of m... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5396490 Mon, 07 Nov 2011 05:00:00 +0100 5396490 http://www.medworm.com/index.php?rid=5374774&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111879 We describe the generation of double knock-in embryonic stem (ES) cells where SPAK and OSR1 cannot be activated by WNK1. We establish that the NKCC1 co-transporter, a proposed target of the WNK pathway, is not phosphorylated or activated in knock-in deficient in SPAK/OSR1 activity. We observe that activity of WNK1 and WNK3 was markedly elevated in the knock-in cells, demonstrating that SPAK/OSR1 significantly influence WNK activity. We show that WNK isoforms interact via a C-terminal coiled-coil domain and identify point mutations of conserved residues within this domain that ablate ability of WNK isoforms to interact. Employing these mutants we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation at least of overexpressed WNK isofor... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5374774 Fri, 28 Oct 2011 04:00:00 +0100 5374774 http://www.medworm.com/index.php?rid=5330243&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111415 The E3 ubiquitin ligase Pellino 1 can be interconverted between inactive and active forms by a reversible phosphorylation mechanism. In vitro, phosphorylation and activation can be catalysed by either the Interleukin-1 Receptor Associated Kinases IRAK1 and IRAK4 or the IkB kinase (IKK)-related kinases (IKKe and TBK1). Here we establish that IRAK1 is the major protein kinase that mediates the interleukin 1 (IL-1)-stimulated activation of Pellino 1 in mouse embryonic fibroblasts (MEFs) or HEK293 cells, while the IKK-related kinases activate Pellino 1 in Tumour Necrosis Factor a (TNFa)-stimulated MEFs. IKKe/TBK1 are also the major protein kinases that activate Pellino 1 in response to Toll-Like Receptor (TLR) ligands that signal via the adaptors MyD88 and/or TRIF. These studies demonstrate th... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5330243 Tue, 18 Oct 2011 04:00:00 +0100 5330243 http://www.medworm.com/index.php?rid=5310544&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111617 This study provides new molecular insight in to the membrane insertion and oligomerization of TPCs. (Source: BJ Signal) BJ Signal journals http://www.medworm.com/rss/comments.php?id=5310544 Wed, 12 Oct 2011 04:00:00 +0100 5310544 http://www.medworm.com/index.php?rid=5292619&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110970 Mitogen and stress activated kinase (MSK) 1 and 2 are nuclear protein kinases that regulate transcription downstream of the ERK1/2 and p38a MAPKs via the phosphorylation of CREB and histone H3. Previous studies on MSKs function have used two inhibitors, H89 and Ro 31-8220, both of which have multiple off target effects. Here we report the characterisation of the in vitro and cellular properties of an improved MSK1 inhibitor, SB-747651A. In vitro, SB-747651A inhibits MSK1 with an IC50 of 11 nM. Screening of an in vitro panel of 117 protein kinases revealed that at 1 mM SB-747651A inhibited 4 other kinases, PRK2, RSK1, p70S6K and ROCK2 with a similar potency to MSK1. In cells, SB-747651A fully inhibited MSK activity at 5 to 10 mM. SB-747651A was found to inhibit the production of the anti-in... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5292619 Wed, 05 Oct 2011 04:00:00 +0100 5292619 http://www.medworm.com/index.php?rid=5281218&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111175 Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilised human platelets and contribute to thrombin-mediated platelet activation. Here we further characterise NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI-specific agonist, collagen-related peptide.. Using a radioligand binding assay, we reveal an NAADP binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases lo... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5281218 Mon, 03 Oct 2011 04:00:00 +0100 5281218 http://www.medworm.com/index.php?rid=5268376&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110967 In conclusion, DHA and pure procyanidins are strong and selective inhibitors of cyclooxygenase activity and NF-κB activation through a p105/p50-dependent regulatory mechanism. (Source: BJ Signal) BJ Signal journals http://www.medworm.com/rss/comments.php?id=5268376 Wed, 28 Sep 2011 04:00:00 +0100 5268376 http://www.medworm.com/index.php?rid=5246009&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111318 We report herein that co-factor-dependent activation of recombinant PKCα was increased by γ-tocopherol and was inhibited by α-tocopherol. Oxidative activation of PKCα was inhibited by α-tocopherol at a 10 fold lower concentration than γ-tocopherol. In binding studies, NBD-tagged-α-tocopherol directly bound to full-length PKCα or the PKCα-C1a domain but not PKCζ. NBD-tagged-α-tocopherol binding to PKCα or the PKCα-C1a domain was blocked by diacylglycerol, α-tocopherol, γ-tocopherol, and retinol but not by cholesterol or phosphatidylserine (PS). Tocopherols enhanced PKCα-C2 domain binding to PS-containing lipid vesicles. In contrast, the PKCα-C2 domain did not bind to lipid ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5246009 Wed, 21 Sep 2011 04:00:00 +0100 5246009 http://www.medworm.com/index.php?rid=5233095&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111264 Intracellular pH conditions many cellular systems but its mechanisms of regulation and perception are mostly unknown. We have identified two yeast genes important for tolerance to intracellular acidification caused by weak permeable acids. One corresponded to LEU2 and functions by removing the dependency of the leu2 mutant host strain on uptake of extracellular leucine. Leucine transport is inhibited by intracellular acidification and either leucine over-supplementation or over-expression of the transporter gene BAP2 improved acid growth. Another acid-tolerance gene is GCN2, encoding a protein kinase activated by uncharged tRNAs during amino acid starvation. Gcn2 phosphorylates eIF2α(Sui2) at Ser-51 and this inhibits general translation but activates that of Gcn4, a transcription fa... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5233095 Thu, 15 Sep 2011 04:00:00 +0100 5233095 http://www.medworm.com/index.php?rid=5233096&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111500 RASSF1A and RASSF1C are two ubiquitously expressed isoforms of RASSF1 gene. The promoter of RASSF1A is frequently hypermethylated, resulting in inactivation in various human cancers. RASSF1A is implicated in the regulation of apoptosis, microtubule stability and cell cycle arrest. However, little is known about the regulation and function of RASSF1C. Here, we show that exogenously expressed RASSF1C is a very unstable protein, which is highly polyubiquitylated and degraded via proteasome. Furthermore, RASSF1C degradation is enhanced when cells are exposed to stress signals, such as UV irradiation. Mule, a HECT family E3 ligase but not SCFb-TrCP and CUL4-DDB1 is RASSF1C E3 ligase under normal condition, while both Mule and SCFβ-TrCP target RASSF1C degradation in response to UV irradia... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5233096 Mon, 12 Sep 2011 04:00:00 +0100 5233096 http://www.medworm.com/index.php?rid=5203891&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111221 Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer’s disease. These modulators bind within the dimer interface of the ligand-binding domain and stabilize the agonist-bound conformation slowing receptor desensitization and/or deactivation. Here, we employ isothermal titration calorimetry to determine binding affinities and thermodynamic details of binding of modulators of GluA2. A mutant of the ligand-binding domain of GluA2 (LBD-L483Y-N754S) that forms a stable dimer in solution was utilized. The potent GluA2 modulator BPAM-97 was used as reference compound. Evidence that BPAM-97 binds in the same pocket as the well-known GluA2 modulator cyclothiazide was obtained from X-ray s... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5203891 Wed, 07 Sep 2011 04:00:00 +0100 5203891 http://www.medworm.com/index.php?rid=5155311&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101980 Skeletal muscle responds to exercise by activation of signaling pathways that coordinate gene expression to sustain muscle performance. MEF2-dependent transcriptional activation of myosin heavy chain (MHC) genes promotes the transformation from fast-twitch into slow-twitch fibers, with MEF2 activity being tightly regulated by interaction with class IIa HDACs. Protein kinase D (PKD) is known to directly phosphorylate skeletal muscle class IIa HDACs mediating their nuclear export and thus derepression of MEF2. Here we report the generation of transgenic mice with inducible conditional expression of a dominant-negative PKD1kd protein in skeletal muscle to assess the role of PKD in muscle function. In control mice, long-term voluntary running experiments resulted in a switch from type IIb &#x0... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5155311 Wed, 17 Aug 2011 23:00:00 +0100 5155311 http://www.medworm.com/index.php?rid=5155310&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110817 Sphingosine 1-phosphate (S1P) is a signaling molecule involved in a host of cellular and physiological functions – most notably cell survival and migration. S1P, which signals via a set of five G protein coupled receptors (S1P1-5), is formed by the action of two sphingosine kinases (SphKs) from sphingosine. Interfering RNA strategies and SphK1 null mouse studies implicate SphK1 in multiple signaling cascades, yet there is a paucity of potent and selective SphK1 inhibitors necessary to evaluate the effects of rapid onset inhibition of this enzyme. We have identified a set of sub-micromolar, amidine-based SphK1 inhibitors and herein report using a pair of these compounds to probe the cellular and physiological functions of SphK1. In doing so, we demonstrate that our inhibitors effecti... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5155310 Wed, 17 Aug 2011 23:00:00 +0100 5155310 http://www.medworm.com/index.php?rid=5155309&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110880 The relative expression patterns of the two insulin receptor isoforms, +/- exon11 (IR-B/A respectively), are tissue dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms, to test whether tissue-preferential biological effects can be attained. In rats and mice, IR-B is the most prominent isoform in liver (<95%) and fat (<90%), whereas in muscles IR-A is the dominant isoform (<95%). As a consequence, insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency (compared to human insulin, HI) for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, whi... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5155309 Wed, 17 Aug 2011 23:00:00 +0100 5155309 http://www.medworm.com/index.php?rid=5155308&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111286 Human aldo-keto reductase 1C proteins (AKR1C1-AKR1C4) exhibit relevant activity with steroids, regulating hormone signaling at pre-receptor level. We here investigate the activity of the four human AKR1C enzymes with retinol and retinaldehyde. All the enzymes except AKR1C2 showed retinaldehyde reductase activity with low Km values (~1 μM). The kcat values were also low (0.18 - 0.6 min−1), except for AKR1C3 with 9‑cis-retinaldehyde whose kcat was remarkably higher (13 min−1). Structural modeling of the AKR1C complexes with 9-cis-retinaldehyde indicated a distinct conformation of Trp-227, originated by changes in residue 226, which may contribute to activity differences. This was partially supported by the kinetics of the AKR1C3 R226P mutant. Retinol/retinaldehyde... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5155308 Wed, 17 Aug 2011 23:00:00 +0100 5155308 http://www.medworm.com/index.php?rid=5129801&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110550 More than 20 years after the description of the two insulin receptor (IR) isoforms, designed IR-A (lacking exon 11) and IR-B (with exon 11) nearly every functional aspect of the alternative splicing both in vitro and in vivo remains controversial. In particular, there is no consensus on the precise ligand binding properties of the isoforms. Increased affinity and dissociation kinetics have been reported for IR-A in comparison to IR-B, but the reverse results have also been reported. These are not trivial issues considering the reported possible increased mitogenic potency of IR-A, and the reported link between slower dissociation and increased mitogenesis. We have re-examined the ligand binding properties of the two isoforms using a novel rigorous mathematical analysis based on the conce... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5129801 Sun, 14 Aug 2011 23:00:00 +0100 5129801 http://www.medworm.com/index.php?rid=5129800&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110871 The early steps in vertebrate vision require fast interactions between rhodopsin (Rho) and transducin (Gt), which are classically described by a collisional coupling mechanism driven by the free diffusion of monomeric proteins on the disc membranes of rod and cone cells. Recent findings, however, point to a very low mobility for Rho and support a substantially different supramolecular organization. Moreover, Rho-Gt interactions seem to possibly occur even prior to light stimuli, which is also difficult to reconcile with the classical scenario. We investigated the kinetics of interaction between native Rho and Gt in different conditions by surface plasmon resonance and analysed the results in the general physiological context by employing a holistic systems modelling approach. Our data po... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5129800 Sun, 14 Aug 2011 23:00:00 +0100 5129800 http://www.medworm.com/index.php?rid=5094104&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110545 In this study, we identified the purified 42 and 35 kDa protein as the heterotrimeric G protein subunit Gαq and Gβ1, respectively. When FLAG-tagged Gαq or Gβ1 was expressed in cells and purified, significant lysoPLD activity was observed in microsomal fractions. Levels of the hydrolyzed product, choline, increased over time, and the Mg2+ dependency and substrate specificity of Gαq were similar to those of lysoPLD purified from the rat brain. Mutation of Gαq at amino acid K52, T186 or D205, residues predicted to interact with nucleotide phosphates or catalytic Mg2+, dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating these substrate binding sites are not identical. Whereas the enzym... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5094104 Tue, 02 Aug 2011 23:00:00 +0100 5094104 http://www.medworm.com/index.php?rid=5048047&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110592 We describe here mutations of the catalytic subunit α of protein kinase A (PKA) that introduce amino acid side chains into the ATP binding site and progressively transform the pocket to mimic that of Aurora protein kinases. The resultant PKA variants are enzymatically active and exhibit high affinity for ATP site inhibitors that are specific for Aurora kinases. These features make the Aurora-chimeric PKA a valuable tool for structure based drug discovery tasks. Analysis of crystal structures of the chimera reveal the roles of individual amino acid residues in the binding of a variety of inhibitors, offering key insights into selectivity mechanisms. Furthermore the high affinity for Aurora kinase specific inhibitors, combined with the favorable crystallizability properties of PKA all... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5048047 Wed, 20 Jul 2011 23:00:00 +0100 5048047 http://www.medworm.com/index.php?rid=5048048&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110230 It is unclear what proportion of a G protein-coupled receptor is present in cells as dimers or oligomers. Saturation BRET studies demonstrated the orexin OX1 receptor to be present in such complexes. Forms of this receptor containing a minimal epitope tag, with the C-terminus linked to YFP or modified at the N-terminus to incorporate a SNAP-tag, migrated in SDS-PAGE as monomers indicating a lack of covalent interactions. Solubilisation with dodecylmaltoside, followed by Blue Native-PAGE, indicated the receptor constructs migrated predominantly as anticipated for dimeric species with evidence for further, higher-order complexes and this was true over a wide range of expression levels. Addition of SDS prior to separation on Blue Native-PAGE resulted in much of the previously dimeric, and all... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5048048 Mon, 18 Jul 2011 23:00:00 +0100 5048048 http://www.medworm.com/index.php?rid=5035324&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102167 We report that Group IVA cytosolic phospholipase A2 (PLA2G4A) is important in the development and function of rodent testes. Interstitial cells of rat testes had high PLA2 activity that was very sensitive to the PLA2G4A-preferential inhibitor AACOCF3. PLA2G4A protein was expressed primarily in interstitial cells of wild-type mouse testes throughout maturation. While Pla2g4a knockout (Pla2g4a-/-) male mice are fertile, their sexual maturation was delayed as indicated by cauda epididymal sperm count and seminal vesicle development. Delayed function of Pla2g4a-/- testes was associated with histological abnormalities including disorganized architecture, swollen appearance, and fewer interstitial cells. Basal secretion of testosterone was significantly attenuated and steroidogenic response to h... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5035324 Thu, 14 Jul 2011 23:00:00 +0100 5035324 http://www.medworm.com/index.php?rid=5026678&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110284 The c-Jun N-terminal kinase (JNK) is part of a Mitogen-activated protein kinase (MAPK) signaling cascade. Scaffold proteins simultaneously associate with various components of the MAPK signaling pathway and play a crucial role in signal transmission and MAPK regulation. WDR62 is a JNK scaffold protein. Recessive mutations within WDR62 result in severe cerebral cortical malformation. Here we demonstrate the association of WDR62 with both JNK2 and the JNK2 activating kinase, MKK7, endogenous and overexpressed proteins. Association of WDR62 with JNK2 and MKK7 occurs via direct protein-protein interactions. We mapped the docking domain of WDR62 responsible for the association with JNK. WDR62 interacts with all JNK isoforms through a D domain motif located at the C-terminus. A WDR62 mutant lack... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5026678 Tue, 12 Jul 2011 23:00:00 +0100 5026678 http://www.medworm.com/index.php?rid=5026677&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110607 Exposure of Saccharomyces cerevisiae to alkaline pH represents a stress condition that generates a compensatory reaction. Here we examine a possible role of the protein kinase-A (PKA) pathway in this response. The phenotypic analysis reveals that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation enhances tolerance to this stress. We observe that alkalinisation causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding to STRE sequences in many promoters. Consequently, a synthetic STRE-LacZ reporter shows a rapid induction in response to alk... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5026677 Tue, 12 Jul 2011 23:00:00 +0100 5026677 http://www.medworm.com/index.php?rid=5017641&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110521 The protein kinase C related kinases (PRKs also referred to as PKNs) are a kinase family important in diverse functions including migration and cytokinesis. Here, we have re-evaluated and compared specificity for PKN1 and PKN3, and assessed predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and 3 phosphorylate serine residues in sequence contexts that have an arginine at position -3. In contrast, PKN1 and 3 do not tolerate Arg in position +1 and -1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP170; a PKN target site was identified that conformed to the predicted pattern. Using a protein array, we identified 22 further s... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5017641 Mon, 11 Jul 2011 23:00:00 +0100 5017641 http://www.medworm.com/index.php?rid=5006158&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101650 The association of the SH3 domain of Src Family Kinases (SFKs) with protein partners bearing proline rich motifs has been implicated in the regulation of the activity, and recently described as a possible mechanism of relocalization to subcellular compartments, of SFKs. We demonstrate for the first time that p13, an accessory protein encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds the SH3 domain of SFKs via its C-terminal proline rich motif, forming a stable heterodimer that translocates to mitochondria by virtue of its N-terminal mitochondrial localization signal. As a result, the activity of SFKs is dramatically enhanced with subsequent increase in mitochondrial tyrosine phosphorylation and the recognized p13’s ability to insert itself into the inner mitochondr... BJ Signal journals http://www.medworm.com/rss/comments.php?id=5006158 Wed, 06 Jul 2011 23:00:00 +0100 5006158 http://www.medworm.com/index.php?rid=4959165&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101581 Sperm PLC-zeta (PLCz) is a distinct phosphoinositide-specific phospholipase C isoform that is proposed to be the physiological trigger of egg activation and embryo development at mammalian fertilization. Recombinant PLCz has the ability to trigger Ca2+ oscillations when expressed in eggs, but it is not known how PLCz activity is regulated in sperm or eggs. Here, we have transfected CHO cells with PLCz fused with either YFP or luciferase and found that PLCz-transfected cells did not display cytoplasmic Ca2+ oscillations any different from control cells. PLCz expression was not associated with changes in CHO cell resting Ca2+ levels, nor in a significantly changed Ca2+ response to extracellular ATP compared to control cells transfected with either YFP alone, a cat... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4959165 Tue, 21 Jun 2011 23:00:00 +0100 4959165 http://www.medworm.com/index.php?rid=4931918&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110502 Genetic alterations in PI 3-kinase signalling are common in cancer and include deletions in PTEN, amplifications of PIK3CA and mutations in two distinct regions of the PIK3CA gene. This suggests drugs targeting PI 3-kinase, and p110α in particular, might be useful in treating cancers. Broad spectrum inhibition of PI 3-kinase is effective in preventing growth factor signalling and tumour growth but suitable inhibitors of p110a have not been available to study the effects of inhibiting this isoform alone. Here, we characterise a novel small molecule, A66, showing the S-enantiomer to be a highly specific and selective p110α inhibitor. Using molecular modelling and biochemical studies we explain the basis of this selectivity. Using a panel of isoform selective inhibitors we show ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4931918 Mon, 13 Jun 2011 23:00:00 +0100 4931918 http://www.medworm.com/index.php?rid=4910204&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110203 We have recently shown that the inter-domain interaction between the two domains of ryanodine receptor (RyR), calmodulin binding domain (CaMBD) and CaM-like domain (CaMLD), activates the Ca2+ channel, and this process is called ‘formation of activation-link’ [Gangopadhyay, J. P. and Ikemoto, N. (2008) Biochem. J. 411 415-423]. Thus, CaM that is bound to the CaMBD is expected to interfere the activation-link formation, thereby stabilizing the closed state of channel under normal conditions. Here we report that upon stimulation of neonatal cardiomyocytes with pro-hypertrophy agonist endothelin-1 (ET-1), CaM dissociates from the RyR, which induces a series of intracellular events: increased frequency of Ca2+ transients, translocation of the signaling molecules CaM,... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4910204 Tue, 07 Jun 2011 23:00:00 +0100 4910204 http://www.medworm.com/index.php?rid=4910205&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110720 Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. Mass spectrometry and bioinformatic analyses estimate that ~ 30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases. However, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as protei... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4910205 Mon, 06 Jun 2011 23:00:00 +0100 4910205 http://www.medworm.com/index.php?rid=4891405&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110351 Cytokines and their receptors regulate hematopoiesis by controlling cellular growth, survival, and differentiation. Thus, it is not surprising that mutations of cytokine receptors contribute to the formation of hematopoietic disorders including cancer. We recently identified transforming properties of the ligand-binding component of the receptor for interleukin-27 (IL27R). While wildtype IL27R exhibits transforming properties in hematopoietic cells, in this study we set out to determine if the transforming activity of IL27R could be enhanced by mutation. We identified three mutations of IL27R that enhance its transforming activity. One of these mutations is a phenylalanine to cysteine mutation at residue 523 (F523C) in the transmembrane domain of the receptor. The two other mutations ident... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4891405 Tue, 31 May 2011 23:00:00 +0100 4891405 http://www.medworm.com/index.php?rid=4839444&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101386 Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of gluco-lipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)-ceramides. Of note, gluco-lipotoxicity specifically induced the formation of 18:0, 22:0 and 24:1 (dihydro)-ceramide molecular species, which was associated with the up-regulation of ceramide synthase 4 (CerS4) levels. Fumonisin-B1, a ceram... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4839444 Wed, 18 May 2011 23:00:00 +0100 4839444 http://www.medworm.com/index.php?rid=4827462&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110479 Calcium homeostasis modulator 1 (CALHM1), a membrane protein with similarity to NMDA receptor channels that localizes in the plasma membrane and the endoplasmic reticulum (ER)of neurons, has been shown to generate a plasma membrane Ca2+ conductance and has been proposed to influence Alzheimer Disease risk. In the present study we have investigated the effects of CALHM1 on intracellular Ca2+ handling in HEK293T cells by using targeted aequorins for selective monitorization of Ca2+ transport by organelles. We find that CALHM1 increases Ca2+ leak of the ER and, more importantly, reduces ER Ca2+ uptake by decreasing both the transport capacity and the Ca2+ affinity of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). As a result, the Ca2&#x00... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4827462 Sun, 15 May 2011 23:00:00 +0100 4827462 http://www.medworm.com/index.php?rid=4819470&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110522 Anopheles gambiae (Agam) relies on its olfactory system to target human prey, leading eventually to injection of Plasmodium falciparum, the malaria vector. Odorant-binding proteins (OBPs) are the first line of proteins involved in odorant recognition. They interact with olfactory receptors and thus constitute an interesting target for insect control. We undertook a large-scale study of proteins belonging to the olfactory system of Agam with the aim of preventing insect bites by designing strong olfactory repellents. We determined the 3D structures of several Agam OBPs alone or in complex with model compounds. Here, we report the first 3D structure of a member of the OBP C+ class, AgamOBP47, which has a longer sequence than classical OBPs and contains 6 disulphide bridges. AgamOBP47 ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4819470 Wed, 11 May 2011 23:00:00 +0100 4819470 http://www.medworm.com/index.php?rid=4811820&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110374 The functional selectivity of adrenergic ligands for activation of beta1 and beta2 adrenoceptor (AR) subtypes has been extensively studied in cAMP signalling. Much less is known about ligand selectivity for arrestin-mediated signalling pathways. Here we used resonance energy transfer methods to compare the ability of beta1 and beta2 adrenoceptors to form a complex with the G proteins beta subunit or beta-arrestin 2 in response to a variety of agonists with varying degrees of efficacy. The profiles of beta1/beta2 AR selectivity of the ligands for the two receptor-transducer interactions were sharply different. For G protein coupling, the majority of ligands were more effective in activating the beta2-AR, whereas for arrestin coupling the relationship was reversed. These data indicate that t... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4811820 Tue, 10 May 2011 23:00:00 +0100 4811820 http://www.medworm.com/index.php?rid=4811821&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102178 Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating diverse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a comparable manner to the unrelated small heat-shock proteins (sHsps). 1H NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilises 14-3-3ζ but has no direct role in chaperone action. K49 is an important functional residue within 14-3-3ζ’s ligand binding groov... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4811821 Mon, 09 May 2011 23:00:00 +0100 4811821 http://www.medworm.com/index.php?rid=4675817&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101031 The c-Jun dimerization protein 2, JDP2, is a member of the basic leucine zipper family of transcription factors that is ubiquitously expressed in all examined cell types. JDP2 is phosphorylated on threonine 148 by the c-Jun N-terminal kinase (JNK) and p38 kinase, although the functional role of its phosphorylation is unknown. Here we show that the JDP2 protein level is dramatically reduced in response to serum stimulation, anisomycin treatment, ultra violet light irradiation, and cycloheximide treatment, all of which activate the JNK pathway. In addition, endogenous and overexpressed JDP2 are phosphorylated in response to these stimuli. Replacement of threonine 148 with alanine stabilizes ectopically expressed JDP2 in the presence of the stimuli; conversely, substitution with glutamic acid... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4675817 Sun, 03 Apr 2011 23:00:00 +0100 4675817 http://www.medworm.com/index.php?rid=4621385&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101791 The EGFR/ErbB/HER family of kinases contains four homologous receptor tyrosine kinases that are important regulatory elements in key signaling pathways. To elucidate the atomistic mechanisms of dimerization-dependent activation in the ErbB family, we have performed molecular dynamics simulations of the intracellular kinase domains of three members of the ErbB family (those with known kinase activity), namely EGFR, ErbB2 (HER2) and ErbB4 (HER4), in different molecular contexts: monomer vs. dimer, wildtype vs. mutant. Using bioinformatics and fluctuation analyses of the molecular dynamics trajectories, we relate sequence similarities to correspondence of specific bond-interaction networks and collective dynamical modes. We find that in the active conformation of the ErbB kinases, key subdoma... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4621385 Tue, 22 Mar 2011 00:00:00 +0100 4621385 http://www.medworm.com/index.php?rid=4610141&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102103 The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth and is a key target for therapeutic intervention in cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1), consisting of mTOR, Raptor and mLST8 and complex 2 (mTORC2) consisting of mTOR, Rictor, Sin1, mLST8 and Protor-1 or Protor-2. Both complexes phosphorylate the hydrophobic motifs of AGC kinase family members: mTORC1 phosphorylates S6K, while mTORC2 regulates phosphorylation of Akt, PKC and SGK1. To investigate the roles of the Protor isoforms, we generated single as well as double Protor-1 and Protor-2 knockout mice and studied how activation of known mTORC2 substrates was affected. We observed that loss of Protor-1 and/or Protor-2 did not affect the expression of ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4610141 Fri, 18 Mar 2011 00:00:00 +0100 4610141 http://www.medworm.com/index.php?rid=4605109&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101201 The hypoxia inducible factor (HIF) plays a central regulatory role in oxygen homeostasis. HIF proteins are regulated by three Fe(II)- and α-ketoglutarate-dependent prolyl hydroxylase enzymes (PHD isozymes 1-3, or PHD1, 2, 3) and one asparaginyl hydroxylase (FIH, factor inhibiting HIF). The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-ketoglutarate-dependent hydroxylases is of great importance. Herein, we report the biochemical characterization of the first HIF protein sub... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4605109 Thu, 17 Mar 2011 00:00:00 +0100 4605109 http://www.medworm.com/index.php?rid=4574540&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102037 The fungal toxin fusicoccin induces plant wilting by affecting ion transport across the plasma membrane of plant cell. The activity of this toxin is so far unknown in humans. Here we show that fusicoccin is able to affect the platelet aggregation process. The toxin associates to platelet intracellular binding sites and induces aggregation in platelet-rich plasma in a dose-dependent manner. We identified the adhesion receptor glycoprotein Ib-IX-V as fusicoccin target. The toxin promotes the binding of the regulatory 14-3-3 proteins to glycoprotein Ibα and hampers that to glycoprotein Ibβ subunit. As a result, platelet adhesion to von Willebrand Factor is stimulated, leading to platelet spreading and integrin αIIbβ3 activation. We anticipate our study to be a ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4574540 Fri, 11 Mar 2011 00:00:00 +0100 4574540 http://www.medworm.com/index.php?rid=4569950&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101360 Protease-activated receptors 1 and 4 belong to the family of G protein-coupled receptors which induce both Gα12/13 and Gαq signalling. By applying the specific PAR1- and PAR4-activating hexapeptides SFLLRN and AYPGKF, respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor-activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca2+ mobilization, protein kinase C signalling, α granules secretion, as well as a complete lack of dense granules secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling-desensitization by differentially reconstituting these affected signalling... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4569950 Thu, 10 Mar 2011 00:00:00 +0100 4569950 http://www.medworm.com/index.php?rid=4516602&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110040 In this study we characterize a next generation Akt inhibitor MK-2206. MK-2206 is an orally active allosteric Akt inhibitor under development for treating solid tumors. We report here that MK-2206 potently inhibits Thr308Akt and Ser473Akt phosphorylation in 3T3-L1 adipocytes (IC50 0.11 mM and 0.18 mM respectively) as well as downstream effects of insulin on GLUT4 translocation (IC50= 0.47 mM) and glucose transport (IC50=0.14 mM). Notably, the potency of MK-2206 is approximately one log higher than previous inhibitors and its specificity is significantly improved with modest inhibitory effects on glucose transport in GLUT4 expressing adipocytes and GLUT1-rich human erythrocytes, independently of Akt. Nevertheless, MK-2206 clearly has potent effects on Akt2, the principal isoform involved in... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4516602 Thu, 24 Feb 2011 00:00:00 +0100 4516602 http://www.medworm.com/index.php?rid=4488661&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101184 Cyclic AMP phosphodiesterase-4 (PDE4) isoforms underpin compartmentalised cAMP signalling in mammalian cells through targeting to specific signalling complexes. Their importance is apparent as PDE4 selective inhibitors exert profound anti-inflammatory effects and act as cognitive enhancers. The p38 MAPK signalling cascade is a key signal transduction pathway involved in the control of cellular immune, inflammatory and stress responses. Here we show that phosphodiesterase-4A5 (PDE4A5) is phosphorylated at Ser147, within the regulatory UCR1 domain conserved amongst PDE4 long isoforms, by the p38 MAPK activated kinase, MK2 (MAPKAPK2). Phosphorylation by MK2, while not altering PDE4A5 activity, markedly attenuates PDE4A5 activation through phosphorylation by protein kinase A (PKA). This modifi... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4488661 Thu, 17 Feb 2011 00:00:00 +0100 4488661 http://www.medworm.com/index.php?rid=4488660&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101517 Sustained over-supply of saturated non-esterified free fatty acids has been shown to promote skeletal muscle insulin resistance, which may be driven, in part, by an increase in inflammatory signalling within this tissue. Here we show that exposure of L6 myotubes to palmitate, a saturated fatty acid, induces activation of the NFkB pathway (based on increased IKK phosphorylation, IkBa loss and elevated IL-6 mRNA expression) and that this was associated with enhanced phosphorylation/activation of p38 MAPK, JNK and ERK as well as impaired insulin-dependent activation of PKB/Akt and glucose transport. NFkB activation by palmitate was unaffected by pharmacological inhibition of p38 MAPK or JNK, but was suppressed significantly by inhibition of MEK/ERK signalling. The importance of ERK with respe... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4488660 Thu, 17 Feb 2011 00:00:00 +0100 4488660 http://www.medworm.com/index.php?rid=4476869&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101726 Connexin 50 (Cx50), a member of the α family of gap junction proteins expressed in the lens of the eye, has been shown to be essential for normal lens development. In the present study, we identified a calmodulin (CaM) binding domain (CaMBD, residues 141-166) in the intracellular loop of Cx50. Elevations in intracellular Ca2+ concentration effected a 95% decline in junctional conductance (gj) of Cx50 in N2A cells that is likely mediated by CaM, because inclusion of the CaM inhibitor, calmidazolium, prevented this Ca2+-dependent decrease in gj. The direct involvement of the Cx50 CaMBD in this Ca2+/CaM-dependent regulation was further demonstrated by inclusion of a synthetic peptide encompassing the CaMBD in both whole cell patch pipettes, which effectively preven... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4476869 Mon, 14 Feb 2011 00:00:00 +0100 4476869 http://www.medworm.com/index.php?rid=4454608&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101749 Adenosine A2A receptors (A2AR) are highly enriched in the striatum, which is the main motor control CNS area. Bioluminescence resonance energy transfer (BRET) assays showed that A2AR homomers may act as cell surface adenosine deaminase (ADA; EC 3.5.4.4) binding proteins. ADA binding affected the quaternary structure of A2AR present on the cell surface. ADA binding to adenosine A2A receptors increased both, agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK 1/2 phosphorylation. Collectively, the results showed that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This p... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4454608 Wed, 09 Feb 2011 00:00:00 +0100 4454608 http://www.medworm.com/index.php?rid=4449632&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101725 CFTR has been shown to form multiple-protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signaling in cells. Targeting protein-protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted PDZ-based LPA2-NHERF2 interaction within the CFTR-NHERF2-LPA2-containing macromolecular complexes at airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits LPA2-NHERF2 interaction. We show that this compound can disrupt LPA2-NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elev... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4449632 Tue, 08 Feb 2011 00:00:00 +0100 4449632 http://www.medworm.com/index.php?rid=4449631&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110013 Neuronal calcium sensor (NCS) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini that could therefore serve as structural control elements providing specific functions like target recognition or Ca2+-sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. We investigated a series of recoverin forms that were mutated at the C-terminus. Using pull down assay, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assay, we demonstrated that trunca... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4449631 Tue, 08 Feb 2011 00:00:00 +0100 4449631 http://www.medworm.com/index.php?rid=4449630&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101752 Activation of neutral sphingomyelinase (N-SMase) is an established part of the response of cytokines such as tumor necrosis factor (TNF). However, it remains unclear which of the currently cloned N-SMase isoforms (nSMase1, 2 and 3) are responsible for this activity. In MCF-7 cells, we find that TNF induces late, but not early increases in N-SMase activity, and that nSMase2 is the primary isoform activated, most likely through post-transcriptional mechanisms. Surprisingly, overexpression of tagged or untagged nSMase3 in multiple cell lines had no significant effect on in vitro N-SMase activity. Moreover, only overexpression of nSMase2, but not nSMase1 or nSMase3, had significant effects on cellular sphingolipid levels, increasing ceramide and decreasing sphingomyelin. Additionally, only siR... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4449630 Tue, 08 Feb 2011 00:00:00 +0100 4449630 http://www.medworm.com/index.php?rid=4445115&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102160 Toll-like receptors (TLRs) provide a mechanism for host defense immune responses. Activated TLRs lead to the recruitment of adaptor proteins to their cytosolic tails, which in turn, promote the activation of interleukin-1 receptor-associated kinases (IRAKs). IRAKs act upon their transcription factor targets to influence the expression of genes involved in the immune response. Toll-interacting protein (Tollip) modulates IRAK function in the TLR signaling pathway. Tollip is multimodular with a conserved C2 domain of unknown function. We found that the Tollip C2 domain preferentially interacts with phosphoinositides, most notably with phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), in a Ca2+-independent manner. However, NMR analysis demons... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4445115 Mon, 07 Feb 2011 00:00:00 +0100 4445115 http://www.medworm.com/index.php?rid=4428367&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101854 The central nervous system regulates neuronal excitability by macromolecular signalling complexes that consist of functionally related proteins, including neurotransmitter receptors, enzymes and scaffolds. The composition of these signal complexes is regulated by post-translational modifications, such as phosphorylation and sumoylation. Here, we searched for proteins interacting with the intracellular C-termini of the metabotropic glutamate receptors mGluR8a and mGluR8b and identified proteins of the sumoylation and neddylation machinery. The Sumo E3-ligases Pias1 and Pias3L interacted strongly with mGluR8b and were co-localized with the E2-conjugating Ubc9, Sumo1 and mGluR8b in cell bodies present in the ganglion cell layer of the mammalian retina. Sumo1 conjugation of K882, present in a ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4428367 Wed, 02 Feb 2011 00:00:00 +0100 4428367 http://www.medworm.com/index.php?rid=4336918&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101437 We present evidence that the PtdIns(3,4,5)P3-binding domain is an unusual hybrid, in which a partial pleckstrin homology consensus sequence is spliced into the phosphatase-like domain. Agonist-dependent activation of the PtdIns 3-kinase pathway in NIH3T3 cells drives translocation of PPIP5K1 from the cytosol to the plasma membrane. We have therefore demonstrated receptor-regulated compartmentalization of inositol pyrophosphate synthesis in mammalian cells. (Source: BJ Signal) BJ Signal journals http://www.medworm.com/rss/comments.php?id=4336918 Tue, 11 Jan 2011 00:00:00 +0100 4336918 http://www.medworm.com/index.php?rid=4313447&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101421 Mammalian Pellino isoforms are phosphorylated by IRAK1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. Here we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDM are stimulated with LPS or poly(I:C). This induction occurs via a TRIF-dependent, IRAK-independent pathway and is prevented by inhibition of the IKK-related protein kinases, TBK1 and IKKε. Pellino 1 is not induced in IRF3-/- BMDM, and is only reduced slightly in type 1 interferon receptor-/- BMDM, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKε as bait, and show that IKKε/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288 and Ser293.... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4313447 Wed, 05 Jan 2011 00:00:00 +0100 4313447 http://www.medworm.com/index.php?rid=4308863&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102012 Insulin sensitivity is critically dependent upon activity of PI 3-kinase and generation of the PtdIns(3,4,5)P3 second messenger. PtdIns(3,4,5)P3 can be broken down to PtdIns(3,4)P2 through the action of the SHIP phosphatases. As PtdIns(3,4)P2 levels peak after those of PtdIns(3,4,5)P3, it has been proposed that PtdIns(3,4)P2 controls a negative feedback loop that down-regulates the insulin and PI 3-kinase network. We previously identified two related adaptor proteins termed TAPP1 and TAPP2 that specifically bind to PtdIns(3,4)P2 through their C-terminal PH domain. To determine whether TAPP1 and TAPP2 play a role in regulating insulin sensitivity, we generated knock-in mice that express normal endogenous levels of mutant TAPP1 and TAPP2 that are incapable of binding PtdIns(3,4)P2. These hom... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4308863 Tue, 04 Jan 2011 00:00:00 +0100 4308863 http://www.medworm.com/index.php?rid=4274810&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101136 We report that the protein is transformed from hexacoordinate to pentacoordinate on binding a lipid molecule. This transformation occurs with the ferric oxidation state of the protein, but not the ferrous state, indicating that this process only occurs under an oxidative environment and may thus be related to redox-liked cell signaling mechanisms. Oleate binds to the protein in a 1:1 stoichiometry and with high affinity (KD= 0.7 μM), however stopped-flow kinetic measurements yield a KD value of 110 μM. The discrepancy between these KD values may be rationalized by recognizing that cytoglobin is a disulfide linked dimer and invoking cooperativity in oleate binding. The lipid-induced transformation of cytoglobin from hexacoordinate to pentacoordinate does not occur with similar... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4274810 Mon, 20 Dec 2010 00:00:00 +0100 4274810 http://www.medworm.com/index.php?rid=4263979&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101244 An improved understanding of the roles of protein kinases in intracellular signaling and disease progression has driven significant advances in protein kinase inhibitor discovery. Peptide inhibitors that target the kinase protein substrate-binding site have continued to attract attention. Here, we describe a novel c-Jun N-terminal Kinase (JNK) inhibitory peptide, PYC71N, which inhibits JNK activity in vitro towards a range of recombinant protein substrates including the transcription factors c-Jun, ATF2, Elk1 and the microtubule regulatory protein DCX. Analysis in cell culture studies confirmed the actions of a cell-permeable version of PYC71 to inhibit c-Jun phosphorylation during acute hyperosmotic stress. The analysis of the in vitro data for the kinetics of this inhibition indicated a ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4263979 Fri, 17 Dec 2010 00:00:00 +0100 4263979 http://www.medworm.com/index.php?rid=4263980&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101628 Ubiquitination of the colony-stimulating factor 3 receptor (CSF3R) occurs after activated CSF3Rs are internalized and reside in early endosomes. CSF3R ubiquitination is crucial for lysosomal routing and degradation. The E3 ligase suppressor of cytokine signalling 3 (SOCS3) has been shown to play a major role in this process. Deubiquitinating enzymes remove ubiquitin moieties from target proteins by proteolytic cleavage. Two of these enzymes, AMSH and UBPY, interact with the general endosomal sorting machinery. Whether deubiquitinating enzymes control CSF3R trafficking from early towards late endosomes is unknown. In the present study, we asked whether AMSH, UBPY or a murine family of deubiquitinating enzymes could fulfil such a role. This DUB family comprises four members (DUB1, DUB1A, DUB... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4263980 Wed, 15 Dec 2010 00:00:00 +0100 4263980 http://www.medworm.com/index.php?rid=4248433&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101210 In this report we demonstrate that E2F1 maintains the ability to interact with pRB in its hyperphosphorylated state (ppRB). This interaction is dependent upon the ‘specific’ E2F1 binding site located in the C-terminal domain of pRB. A unique region of the marked box domain of E2F1 contacts the ‘specific’ site to mediate the interaction with ppRB. The mechanistic basis of the interaction between E2F1 and ppRB is subtle. A single substitution between valine and proline in the marked box distinguishes E2F1’s ability to interact with ppRB from the inability of E2F3 to bind to the ‘specific’ site in ppRB. The E2F1-pRB interaction at the ‘specific’ site also maintains the ability to regulate the transcriptional activation of E2F1 tar... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4248433 Fri, 10 Dec 2010 00:00:00 +0100 4248433 http://www.medworm.com/index.php?rid=4232513&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101878 Receptors of the Frizzled (Fz) family initiate Wnt ligand-dependent signaling controlling multiple steps in organism development and carcinogenesis. Fz proteins possess seven transmembrane domains and their signaling depends on heterotrimeric G proteins in various organisms. However, Fzs constitute a distinct group within the G protein-coupled receptors (GPCR) superfamily, and Fz signaling can be G protein-independent in some experimental setups, leading to concerns about the GPCR nature of these proteins. Here we demonstrate that mammalian Fzs act as GPCRs on heterotrimeric Go/i proteins. Addition of the Wnt3a ligand to rat brain membranes or cultured cells elicits Fz-dependent guanine nucleotide exchange on Go/i. These responses were sensitive to a Wnt antagonist and to pertussis toxin, ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4232513 Mon, 06 Dec 2010 00:00:00 +0100 4232513 http://www.medworm.com/index.php?rid=4199967&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101738 Caspase-8 is an initiator caspase that is activated by death receptors to initiate the extrinsic pathway of apoptosis. Caspase-8 activation involves dimerization and subsequent interdomain autoprocessing of caspase-8 zymogens, and recently published work has established that elimination of the autoprocessing site of caspase-8 abrogates its pro-apoptotic function while leaving its proliferative function intact. The observation that the developmental abnormalities of caspase-8 deficient mice are shared by mice lacking the dimerization adapter FADD or the paracaspase FLIPL has led to the hypothesis that FADD-dependent formation of heterodimers between caspase-8 and FLIPL could mediate the developmental role of caspase-8. Using an inducible dimerization system we demonstrate that cleav... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4199967 Thu, 25 Nov 2010 00:00:00 +0100 4199967 http://www.medworm.com/index.php?rid=4178499&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101732 Phosphoinositide-dependent protein kinase-1 (PDK1) activates a group of protein kinases belonging to the AGC-kinase family that play important roles in mediating diverse biological processes. Many cancer-driving mutations induce activation of PDK1 targets including Akt, S6K and SGK. Here we describe the small molecule GSK2334470, which inhibits PDK1 with an IC50 of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 to HEK293, U87 or fibroblast cells ablated T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF1. GSK2334470 also inhibited T-loop phosphorylation and activation of Akt, but was more efficient at inhibiting Akt in r... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4178499 Thu, 18 Nov 2010 00:00:00 +0100 4178499 http://www.medworm.com/index.php?rid=4035575&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100247 In this study, we report a regulatory subunit of protein phosphatase 5 (PP5), G4-1, that physically interacts with IKKβ and negatively regulates NF-κB activation. The association of G4-1 to IKKβ depends on the kinase activity of IKKβ. Mapping of the G4-1 binding domain of IKKβ reveals that the serine-rich domain in the carboxyl terminus of IKKβ is required for G4-1 binding. When seven auto-phosphorylated serine residues in this domain were mutated to alanine, the mutant form of IKKβ lost its ability to bind G4-1 and was more potent than the wild type kinase to activate NF-κB. Knockdown of G4-1 enhanced TNF-α-induced NF-κB activity, and Knockdown of PP5 totally abolished the inhibitory activity of G4-1 on NF-κB act... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4035575 Tue, 05 Oct 2010 23:00:00 +0100 4035575 http://www.medworm.com/index.php?rid=4035576&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101287 The poorly characterised G protein-coupled receptor GPR35 has been suggested as a potential exploratory target for the treatment of both metabolic disorders and hypertension. It has also been indicated to play an important role in immune modulation. A major impediment to validation of these concepts and further study of the role of this receptor has been a paucity of pharmacological tools that interact with GPR35. Using a receptor-β-arrestin 2 interaction assay with both human and rat orthologues of GPR35 we identified a number of compounds possessing agonist activity. These included the previously described ligand zaprinast. Although a number of active compounds, including cromolyn disodium and dicumarol, displayed similar potency at both orthologues of GPR35, a number of ligands, ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=4035576 Mon, 04 Oct 2010 23:00:00 +0100 4035576 http://www.medworm.com/index.php?rid=3997817&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100717 The sodium/proton exchanger NHE1 is a highly regulated membrane protein that is required for pH homeostasis in cardiomyocytes. The activation of NHE1 leads to proton extrusion, which is essential for counteracting cellular acidity that occurs following increased metabolic activity or ischemia. The activation of NHE1 intrinsic catalytic activity has been well characterised and established experimentally. However, we have examined here whether a net translocation of NHE1 to the sarcolemma of cardiomyocytes may also be involved in the activation process. We have determined the distribution of NHE1 by means of immunofluorescence microscopy and cell-surface biotinylation. We have discovered changes in the distribution of NHE1 that occur when cardiomyocytes are stimulated with insulin that are P... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3997817 Thu, 23 Sep 2010 23:00:00 +0100 3997817 http://www.medworm.com/index.php?rid=3997816&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100774 Recent work has highlighted roles for Janus kinase (JAK) family members in hematopoietic diseases. While sequencing efforts have uncovered transforming mutations of JAK1 in acute leukemia, they have also identified non-transforming JAK1 mutations. Thus, with limited knowledge of the mechanisms of JAK1 activation by mutation, sequencing may not readily identify transforming mutations. Therefore, we sought to further understand the repertoire of transforming mutations of JAK1. We identified seven randomly generated transforming JAK1 mutations, including: Val658Leu and a deletion of amino acids 629/630 in the pseudokinase domain, as well as Leu910Pro, Phe938Ser, Pro960Ser, Lys1026Glu, and Tyr1035Cys within the kinase domain. These mutations led to differential signaling activation, but exhibi... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3997816 Thu, 23 Sep 2010 23:00:00 +0100 3997816 http://www.medworm.com/index.php?rid=3942152&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101188 Mammalian Protein Kinase D (PKD) isoforms have been implicated in the regulation of diverse biological processes in response to diacylglycerol and Protein Kinase C (PKC) signalling. To compare the functions of PKD1 and PKD2 in vivo we generated mice deficient in either PKD1 or PKD2 enzymatic activity, via homozygous expression of PKD1Ser744Ala/Ser748Ala or PKD2Ser707Ala/Ser711Ala ‘knockin’ alleles. We also examined PKD2-deficient mice generated using ‘gene trap’ technology. We demonstrate that unlike PKD1, PKD2 catalytic activity is dispensable for normal embryogenesis. We also show that PKD2 is the major PKD isoform expressed in lymphoid tissues but that PKD2 catalytic activity is not essential for the development of mature, peripheral T and B lymphocytes. PKD2... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3942152 Mon, 06 Sep 2010 23:00:00 +0100 3942152 http://www.medworm.com/index.php?rid=3938011&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101010 We have identified the Protein Kinase C (PKC) anchoring protein, Receptor for Activated C Kinase 1 (RACK1), as a specific binding partner for the cyclic AMP-specific phosphodiesterase, PDE4D5, suggesting a potential site for cross-talk between the PKC- and cyclic AMP-signalling pathways. Here we find that elevation of intracellular cyclic AMP with the b2-adrenoceptor agonist, isoproterenol, led to activation of PDE4 enzymes in the particulate and soluble fractions of HEK293 cells, whereas activation of PDE4D5 by isoproterenol and the PKC-activator, PMA, was restricted to the particulate fraction where it interacts with RACK1, however RACK1 is dispensable for anchoring PDE4D5 to the particulate fraction. Kinetic studies did demonstrate that RACK1 alters the conformation of particulate-assoc... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3938011 Sun, 05 Sep 2010 23:00:00 +0100 3938011 http://www.medworm.com/index.php?rid=3932522&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100799 F-box WD40 Protein 7 (Fbw7) is a major tumor suppressor, which mediates the degradation of several potent oncogenes. Protein Kinase C (PKC) is a serine/threonine kinase which can promote transformation when dysregulated. In the present study, we investigated the relationship between Fbw7 and PKC. Multiple members of the PKC superfamily interact with the substrate-binding domain of Fbw7. However, we find no evidence for Fbw7-mediated degradation of PKC. Instead, we demonstrate that Fbw7 is a novel substrate for PKC. Two residues within the isoform specific, N-terminus of Fbw7-α are phosphorylated in a PKC-dependent manner, both in vitro and in mammalian cells (S10 and S18). Mutational analyses reveal that phosphorylation of Fbw7-α at S10 can regulate its nuclear localisation. ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3932522 Thu, 02 Sep 2010 23:00:00 +0100 3932522 http://www.medworm.com/index.php?rid=3932521&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100663 SYNOPSIS Hexokinase 2 (Hxk2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization; it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. To get more insights into structure-function relationships of the Hxk2 protein, we followed two different approaches. In the first we deleted the last eight amino acids of Hxk2 and substituted serine 304 with phenylalanine to generate Hxk2wca. Analysis of this mutant demonstrated that these domains play an essential role in the catalytic activity of yeast Hxk2 but has no effect on the regulatory function of this protein. In the second we analysed whether amino acids fro... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3932521 Thu, 02 Sep 2010 23:00:00 +0100 3932521 http://www.medworm.com/index.php?rid=3861335&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101024 S6K1 is activated by insulin and growth factors via the PI3K and mTOR signalling pathways. S6K1 regulates numerous processes such as protein synthesis, growth, proliferation and longevity and its inhibition has been proposed as a strategy for the treatment of cancer and insulin resistance. Here we describe a novel cell-permeable inhibitor of S6K1, PF-4708671, which specifically inhibits the S6K1 isoform with a Ki of 20 nM and IC50 of 160 nM. PF-4708671 prevents the S6K1-mediated phosphorylation of S6 protein in response to IGF1 while having no effect upon the TPA-induced phosphorylation of substrates of the highly related RSK and MSK kinases. PF-4708671 was also found to induce phosphorylation of the T-loop and hydrophobic motif of S6K1, an effect that is dependent upon mTORC1. PF-4708671 ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3861335 Wed, 11 Aug 2010 23:00:00 +0100 3861335 http://www.medworm.com/index.php?rid=3861334&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100558 A sole Epstein-Barr virus (EBV)-encoded protein kinase (the BGLF4 gene product) plays important roles in viral infection. While a number of targets of this protein have been identified, the kinase itself remains largely unstudied in regard to its enzymology and structure. Here, site-directed mutangenesis has been employed to generate mutations targeting residues involved in nuclear localization of the EBV-PK, core residues in subdomain III of the protein kinase domain conserved in most protein kinases, or residues in subdomain VIa conserved only within herpesvirus-encoded protein kinases (HPKs) group. Deletion of amino acids 389-391 resulted in exclusive cytoplasmic localization of the protein indicating the involvement of this region in nuclear translocation of the EBV-PK. Mutations at th... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3861334 Wed, 11 Aug 2010 23:00:00 +0100 3861334 http://www.medworm.com/index.php?rid=3857620&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101100 In conclusion, contraction causes site-specific phosphorylation of TBC1D1 in skeletal muscle, and TBC1D1 phosphorylation on AMPK sites regulates contraction-stimulated glucose uptake. AMPK and Akt regulate TBC1D1 phosphorylation, but there must be additional upstream kinases that mediate TBC1D1 phosphorylation in skeletal muscle. (Source: BJ Signal) BJ Signal journals http://www.medworm.com/rss/comments.php?id=3857620 Tue, 10 Aug 2010 23:00:00 +0100 3857620 http://www.medworm.com/index.php?rid=3826437&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101089 Based on transfection experiments using a dominant-negative approach, our previous studies suggested that protein kinase B (PKB) was not involved in heart 6-phosphofruco-2-kinase (PFK-2) activation by insulin. Therefore, we first tested whether the serum- and glucocorticoid-inducible protein kinase-3 (SGK3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [γ-32P] labelled ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in HEK 293T cells co-transfected with SGK3 siRNA and heart PFK-2, insulin-induced heart PFK-2 activation was unaffected. The involvement of PKB in heart PFK-2 activation by insulin was re-evaluated using different models: (i) hearts from transgenic mice with a muscle/heart-specific mutation in... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3826437 Wed, 04 Aug 2010 23:00:00 +0100 3826437 http://www.medworm.com/index.php?rid=3783674&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100637 Intracellular Ca2+-dependent cellular responses are often mediated by the ubiquitous protein calmodulin (CaM), which, upon binding Ca2+, can interact with and alter the function of numerous proteins. Here, using a newly developed functional proteomic screen of rat brain extracts, we identified the plasticity related gene-1 (PRG-1) as a novel CaM target. A CaM-overlay, and an immunoprecipitation assay, revealed that PRG-1 is capable of binding the Ca2+/CaM complex in vitro and in transfected cells. Surface plasmon resonance and zero-length crosslinking showed that the C-terminal putative cytoplasmic domain (residues 466-766) of PRG-1 binds equimolar amounts of CaM in a Ca2+-dependent manner, with a relatively high affinity (KD for Ca2+/CaM of 8 nM). Variou... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3783674 Thu, 22 Jul 2010 23:00:00 +0100 3783674 http://www.medworm.com/index.php?rid=3775731&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100784 The Leucine-Rich Repeat Protein Kinase 2 (LRRK2) is mutated in a significant number of Parkinson’s disease patients. Since a common mutation that changes Gly2019 to Ser enhances kinase catalytic activity, small molecule LRRK2 inhibitors might have utility in treating Parkinson’s disease. However, the effectiveness of inhibitors is difficult to assess, as no physiological substrates or downstream effectors have been identified that could be exploited to develop a robust cell-based assay. We recently established that LRRK2 bound 14-3-3 isoforms via its phosphorylation of Ser910 and Ser935. Here we demonstrate that treatment of Swiss 3T3 cells or lymphoblastoid cells derived from control or a Parkinson’s disease patient harbouring a homozygous LRRK2[G2019S] mutation, with... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3775731 Tue, 20 Jul 2010 23:00:00 +0100 3775731 http://www.medworm.com/index.php?rid=3766678&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100483 The Leucine-Rich Repeat Protein Kinase 2 (LRRK2) is mutated in a significant number of Parkinson’s disease patients, but still little is understood about how it is regulated or functions. Here we demonstrate that 14-3-3 isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We also establish that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our data suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity but induces LRRK2 to accumulate within discrete cytoplasmic pools perhaps resembling inclusion bodi... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3766678 Sun, 18 Jul 2010 23:00:00 +0100 3766678 http://www.medworm.com/index.php?rid=3757176&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100383 We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome based on a capped tri-peptide first identified by high-throughput screening of a library of ~350,000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the β5 (chymotrypsin-like) site over the β1 (caspase-like) and β2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds sho... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3757176 Wed, 14 Jul 2010 23:00:00 +0100 3757176 http://www.medworm.com/index.php?rid=3705959&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100341 The beta-isoform of phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinase (PIP4K) regulates the levels of nuclear PtdIns5P, which in turn modulates the acetylation of the tumour suppressor p53. The crystal structure of PIP4Kbeta demonstrated that it can form a homodimer with the two subunits arranged in opposite orientations. Using mass spectrometry, isoform specific antibodies to PIP4Ks, RNAi suppression and overexpression studies, we show that PIP4Kbeta interacts in vitro and in vivo with the PIP4Kalpha isoform. As the two isoforms phosphorylate the same substrate to generate the same product, the interaction could be considered to be functionally redundant. However, contrary to expectation, we find that PIP4Kbeta has 2000 times less activity towards PtdIns5P, compared to PIP4Kalpha and t... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3705959 Sun, 27 Jun 2010 23:00:00 +0100 3705959 http://www.medworm.com/index.php?rid=3694723&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100026 Nucleoside diphosphate kinase (NDK) is primarily involved in maintaining cellular nucleotide pools in both prokaryotes and eukaryotes. We cloned ndk from Salmonella typhimurium and expressed in Escherichia coli as a histidine-tagged protein. The Ni-NTA purified protein (sNDK) was found to be tetrameric with monomeric unit molecular mass of ~18 kDa. The sNDK exhibited divalent cation dependent autophosphorylation at wide range of pH and the phosphorylation withstands acid or alkali treatment. Surprisingly, nucleoside diphosphates did not behave as ‘true inhibitors’ of autophosphorylation activity. The sNDK displayed phospho-transfer activity from nucleoside triphosphates to nucleoside diphosphates, however, it was Mg2+/Mn2+dependent. Mutational analysis establish... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3694723 Wed, 23 Jun 2010 23:00:00 +0100 3694723 http://www.medworm.com/index.php?rid=3690318&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100340 Phosphatidylinositol 5-phosphate 4-kinases (PtdIns5P 4-kinases) IIα and IIβ are respectively cytosolic or nuclear when transfected into cells, including DT40 cells (Richardson et al Cellular Signalling 19, 1309-1314; 2007). Here we have genomically tagged both PtdIns5P 4-kinase II isoforms in DT40 cells, and immunoprecipitation of either isoform from tagged cells followed by mass spectrometry showed that they are associated directly with each other, most likely by heterodimerisation. We quantified the cellular levels of the PtdIns5P 4-kinase II mRNAs by real-time quantitative PCR, and the absolute amount of each isoform in immunoprecipitates by mass spectrometry using selective reaction monitoring with 14N13C-labelled internal standard peptides. The data suggest that... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3690318 Tue, 22 Jun 2010 23:00:00 +0100 3690318 http://www.medworm.com/index.php?rid=3588497&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091649 Pyrroloquinoline quinone (PQQ) improves energy utilization and reproductive performance when added to rodent diets devoid of PQQ. Herein, changes are described in gene expression patterns and transcriptional networks that respond to dietary PQQ restriction or pharmacologic administration. Rats were fed diets either deficient in (PQQ-) or supplemented with (~ 6 nmol PQQ/g of diet, PQQ+) PQQ. In addition, groups of rats were either repleted by administering PQQ to PQQ- rats (1.5 mg PQQ i.p. /kg BW at 12 h intervals for 36 hours) or partially depleted by feeding the PQQ- diet to PQQ+ rats for 48 hours. RNA extracted from liver and a Codelink® UniSet Rat I Bioarray system were used to assess gene transcript expression. Of the ~10000 rat sequences and control probes anal... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3588497 Thu, 20 May 2010 23:00:00 +0100 3588497 http://www.medworm.com/index.php?rid=3551637&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090819 The kinase-related protein, KRP, also known as telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the regulatory light chains of myosin (rMLC) by the Ca2+-activated myosin light chain kinase (MLCK). Using the phosphatase inhibitor, microcystin, we now show that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP depleted triton skinned taenia coli with microcystin at pCa <8 induced a slow contraction reaching 90% of maximal force (Fmax) at pCa 4.5 after ~25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of Fmax was increased from 8 min to 35 min. KRP similar... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3551637 Mon, 10 May 2010 23:00:00 +0100 3551637 http://www.medworm.com/index.php?rid=3529130&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100166 Phosphoinositide (PI)-3 kinases play a critical role in platelet functional responses. PI-3 kinases are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or with Gz, or super-stimulation of Gi pathways. In the current study, we evaluated the role of specific PI-3 kinase isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation, and ERK activation. Our results show that loss of PI-3 kinase signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation, and thromboxane A2 generation. We also show that Gq+Gi- or Gi+Gz-mediated platelet aggregati... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3529130 Mon, 03 May 2010 23:00:00 +0100 3529130 http://www.medworm.com/index.php?rid=3462972&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091660 Signalling through the interferon-α and T cell receptors (IFNαR and TCR respectively) in T lymphocytes results in distinct immune responses. Despite this both receptors elicit ERK MAP kinase phosphorylation. Vav and Slp76 are shown to be required for IFNα-stimulated ERK activity. These form a subset of proteins which behave identically on stimulation of both receptors. TCR deletion abrogates IFNαR-stimulated MAP kinase activity whilst the canonical JAK/STAT pathway is unaffected. Thus, recruitment of the intact TCR early signalling complex (ESC) is necessary for this downstream MAP kinase response. Despite using a common ESC, stimulation of the IFNαR does not produce the transcriptional response associated with TCR. Up-regulation of the MAP kinase pathway... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3462972 Mon, 12 Apr 2010 23:00:00 +0100 3462972 http://www.medworm.com/index.php?rid=3453055&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091864 Acidic phosphoproteins of mineralized tissues such as bone and dentin are believed to play important roles in hydroxyapatite (HA) nucleation and growth. Bone sialoprotein (BSP) is the most potent known nucleator of HA, an activity that is thought to be dependent on phosphorylation of the protein. The present study identifies the role phosphate groups play in mineral formation. Recombinant BSP and peptides corresponding to residues 1-100 and 133-205 of the rat sequence were phosphorylated with protein kinase CK2 (CK2). Phosphorylation increased the nucleating activity of BSP and BSP(133-205) but not BSP(1-100). Mass spectrometry analysis revealed that the major site phosphorylated within BSP(133-205) was Ser136, a site adjacent to the series of contiguous Glu residues previously implicated ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3453055 Thu, 08 Apr 2010 23:00:00 +0100 3453055 http://www.medworm.com/index.php?rid=3448135&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100143 Inositol 1,4,5-trisphosphate receptors (IP3R) are the intracellular channels that mediate release of Ca2+ from the endoplasmic reticulum in response to the many stimuli that evoke IP3 formation. We characterized and purified type 1 IP3R heterologously expressed in Sf9 cells, and used the purified IP3R1 to determine its three-dimensional structure by electron microscopy and single particle analysis. Recombinant IP3R1 has four-fold symmetry with overall dimensions of about 19.5 x 19.5 x 17.5 nm3. It comprises a small domain, which is likely to include the pore, linked by slender bridges to a large cytoplasmic domain with four petal-like regions. Our structures of recombinant IP3R1 and native cerebellar IP3R have similar appearances and dimensions. The only notable difference is the ab... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3448135 Wed, 07 Apr 2010 23:00:00 +0100 3448135 http://www.medworm.com/index.php?rid=3410830&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100139 Two G protein coupled receptors (GPCRs), the thyrotropin-releasing hormone receptor (TRHR) and beta2-adrenergic receptor (beta2AR), are regulated in distinct manners. Following agonist binding, TRHR undergoes rapid phosphorylation attributable to G protein-coupled receptor kinases (GRKs); beta2AR is phosphorylated by both second messenger-activated protein kinase A (PKA) and GRKs with slower kinetics. TRHR co-internalizes with arrestin while beta2AR recruits arrestin but internalizes without it. Both receptors are dephosphorylated following agonist removal, but TRHR is dephosphorylated much faster while it remains at the plasma membrane. We generated chimeras swapping the C-terminal domains of these receptors to clarify the role of different receptor regions in phosphorylation, internaliza... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3410830 Fri, 26 Mar 2010 00:00:00 +0100 3410830 http://www.medworm.com/index.php?rid=3321596&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091672 Enzymes from the PDE4 cAMP-specific phosphodiesterase family are crucial for the maintenance of compartmentalized cAMP responses in many cell types. Regulation of phosphodiesterase (PDE) activity can be achieved via post-translational modification such as phosphorylation by ERK MAP kinases and Protein Kinase A. In the present study we report for the first time that PDE4 isoforms from the PDE4A and PDE4D sub-families can be selectively modified by the small ubiquitin related modifier, SUMO. We identify a single SUMO site within a consensus tetrapeptide motif, y-K-X-E (y represents a hydrophobic residue), which lies in the catalytic unit of these enzymes. SUMO modification of PDE4 at this site was observed upon overexpression of the SUMO E3 ligase, PIASy in HEK cells and we identify PIASy as... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3321596 Tue, 02 Mar 2010 00:00:00 +0100 3321596 http://www.medworm.com/index.php?rid=3275325&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091077 Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK pathways, including the JNK pathway. Immunoblotting of lysates from cells ectopically expressing active MLK3 revealed an additional immunoreactive band corresponding to a carboxyl-terminal fragment (CTF) of MLK3. Herein we provide evidence that MLK3 undergoes proteolysis to generate a stable CTF in response to different stimuli including phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor (TNF) α. The cleavage site was deduced by Edman sequencing as between Gln 251 and Pro 252 which is within the kinase domain of MLK3. Based on our homology model of the kinase domain of MLK3, the region containing the cleavage site is predicted to reside on a flexible, s... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3275325 Tue, 16 Feb 2010 00:00:00 +0100 3275325 http://www.medworm.com/index.php?rid=3275326&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091011 In this study, we identify the actin-binding protein-280 (Filamin A) as a presumed “binder” protein that can bind to MKK7 as well as to MKK4, connecting them in close proximity. We show that Filamin family members A, B and C interact with MKK4 and MKK7 but not with JNK. Filamin A binds to an N-terminal region (residues 31-60) present in the MKK7γ and MKK7β splice isoforms but cannot bind to MKK7α, which lacks these amino acids. This same N-terminal region is crucial for the intracellular colocalization of MKK7γ with actin stress fibers and Filamin A. Experiments using Filamin A deletion mutants revealed that the MKK7-binding region of Filamin A differs from its MKK4-binding region, and that MKK7γ (but not MKK7α) can form a complex wit... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3275326 Mon, 15 Feb 2010 00:00:00 +0100 3275326 http://www.medworm.com/index.php?rid=3243110&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091951 Erythropoietin (EPO), the major hormone regulating erythropoiesis functions via activation of its cell surface receptor (EPO-R) present on erythroid progenitor cells. One of the most striking properties of EPO-R is its low expression on the cell surface, as opposed to its high intracellular levels. The low cell surface expression of EPO-R may thus limit the efficacy of EPO that is routinely used to treat primary and secondary anemia. In a recent study (Nahari et al., Biochem. J. 2008) we have shown that insertion of an NPVY sequence into the intracellular domain of EPO-R increases its cell surface expression. Here we demonstrate that this NPVY EPO-R insert has a selective effect on EPO mediated downstream signaling in Ba/F3 cells expressing this receptor (NPVY-EPO-R). This is monitored by ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3243110 Fri, 05 Feb 2010 00:00:00 +0100 3243110 http://www.medworm.com/index.php?rid=3215761&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091589 Oxidative stress was previously implicated in regulation of ceramide metabolism. Here, its effects on dihydroceramide desaturase were investigated. To stimulate oxidative stress HEK293, MCF7, A549, and SMS-KCNR cells were treated with hydrogen peroxide, menadione, or tert-butylhydroperoxide. In all cell lines, an increase in dihydroceramide was observed upon oxidative stress as measured by LC/MS. In contrast, total ceramide levels were relatively unchanged. Mechanistically, dihydroceramide desaturase activity was measured by an in-situ assay and was decreased in a time- and dose-dependent fashion. Interestingly, no detectable changes in the protein levels were observed, suggesting that oxidative stress does not induce degradation of dihydroceramide desaturase. In summary, oxidative stress ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3215761 Wed, 27 Jan 2010 00:00:00 +0100 3215761 http://www.medworm.com/index.php?rid=3211617&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091768 In conclusion, L. pneumophila induces mitochondria-mediated T cell apoptosis through inhibition of the Akt/GSK-3β signaling pathway. (Source: BJ Signal) BJ Signal journals http://www.medworm.com/rss/comments.php?id=3211617 Tue, 26 Jan 2010 00:00:00 +0100 3211617 http://www.medworm.com/index.php?rid=3207801&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091038 Interleukin (IL)-1β increased granulocyte/macrophage colony-stimulating factor (GM-CSF) expression from pulmonary A549 cells and primary human bronchial epithelial (HBE) cells. These responses were repressed by the glucocorticoid, dexamethasone, allowing use of A549 cells as a relevant model. IL-1β induced GM-CSF release into the culture medium by 6 h and in cell lysates (cytosolic) at 2 h. These were profoundly inhibited by dexamethasone, yet IL-1β-induced GM-CSF mRNA and unspliced nuclear RNA (a surrogate of transcription rate) were modestly inhibited by dexamethasone at times up to 2 h. While this indicates an effect on protein synthesis, actinomycin D chase experiments also indicated post-transcriptional repression by dexamethasone. Dexamethasone-dependent mRNA rep... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3207801 Tue, 26 Jan 2010 00:00:00 +0100 3207801 http://www.medworm.com/index.php?rid=3110598&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091909 Maintenance of cation homeostasis is a key process for any living organism. Specific mutations in Glc7, the essential catalytic subunit of yeast protein phosphatase 1, result in salt and alkaline pH sensitivity, suggesting a role for this protein in cation homeostasis. We screened a collection of Glc7 regulatory subunit mutants for altered tolerance to diverse cations (Na+, Li+, Ca+2) and alkaline pH. Among eighteen candidates, only deletion of REF2 yielded increased sensitivity to these conditions, as well as to diverse organic toxic cations. A Phe-374 to Ala mutated version of Ref2, which cannot bind Glc7, is unable to rescue the salt-related phenotypes of the ref2 strain, suggesting that Ref2 function in cation homeostasis is mediated by Glc7. The ref2 deletion muta... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3110598 Tue, 22 Dec 2009 00:00:00 +0100 3110598 http://www.medworm.com/index.php?rid=3078576&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091266 Pyruvate carboxylase is an enzyme of the so-called pyruvate cycling pathways that have been proposed to contribute to glucose-stimulated insulin secretion in pancreatic beta cells. In the rat insulinoma cell line 832/13, transcripts from both the distal and proximal gene promoter for pyruvate carboxylase are upregulated by glucose, with pyruvate carboxylase being expressed mainly from the distal gene promoter. At position -408/-392 relative to the transcription start site, the distal gene promoter was found to contain a carbohydrate response element (ChoRE). Its deletion abolishes glucose responsiveness of the promoter, and the sequence can mediate glucose responsiveness to a heterologous gene promoter. The carbohydrate response element binding protein (ChREBP) and its dimerization partner... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3078576 Thu, 10 Dec 2009 00:00:00 +0100 3078576 http://www.medworm.com/index.php?rid=3022407&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090615 Janus kinase 2 (JAK2) is important for signaling through many cytokine receptors, and a gain-of-function JAK2 mutation in its pseudokinase domain, V617F, has been implicated in Philadelphia chromosome-negative myeloproliferative neoplasms. How this mutation hyperactivates JAK2 is poorly understood. Here we report our findings that the V617F mutation has little effect on the Vmax of JAK2 kinase activity but lowers the Km value for substrates. Therefore, under physiological conditions where the concentration level of substrates is presumably below saturation, JAK2(V617F) exhibits hyperactivation compared to wild-type JAK2. This lower Km of JAK2(V617F) towards substrates requires the JAK2 FERM domain, as deletion of the FERM domain abolished this effect. We also show that in contrast to its p... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3022407 Tue, 24 Nov 2009 00:00:00 +0100 3022407 http://www.medworm.com/index.php?rid=3011641&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091580 Insulin stimulates endothelial nitric oxide synthesis via protein kinase B/Akt-mediated phosphorylation and activation of endothelial nitric oxide synthase at Ser1177. In previous studies, we have demonstrated that stimulation of endothelial nitric oxide synthase phosphorylation at Ser1177 may be required, yet is not sufficient for insulin-stimulated nitric oxide synthesis. We therefore investigated the role of phosphorylation of endothelial nitric oxide synthase at alternative sites to Ser1177 as candidate parallel mechanisms contributing to insulin-stimulated nitric oxide synthesis. Stimulation of human aortic endothelial cells with insulin rapidly stimulated phosphorylation of both Ser615 and Ser1177 on endothelial NO synthase, whereas phosphorylation of Ser114, Thr495 and Ser633 was un... BJ Signal journals http://www.medworm.com/rss/comments.php?id=3011641 Fri, 20 Nov 2009 00:00:00 +0100 3011641 http://www.medworm.com/index.php?rid=2926774&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091136 We recently reported that the dual-specific A kinase anchoring protein (AKAP) Ezrin targets type I protein kinase A (PKA) to the vicinity of the T cell receptor (TCR) in T cells and together with phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG) and Ezrin/Radixin/Moesin (ERM) binding phosphoprotein 50 (EBP50) forms a scaffold that positions PKA close to its substrate the C-terminal Src kinase (Csk). This complex is important for controlling the activation state of the T cell. Ezrin binds to the adaptor protein EBP50 which again contacts PAG. In this work we show that Ezrin and EBP50 interact with high affinity (KD = 58 ± 7 nM). A peptide corresponding to the Ezrin binding (EB) region in EBP50 (EBP50pep) is used to further characterize the binding ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2926774 Mon, 26 Oct 2009 00:00:00 +0100 2926774 http://www.medworm.com/index.php?rid=2926773&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090380 PKNs form a subfamily of the AGC serine/threonine protein kinases, and have a catalytic domain homologous to that of protein kinase C (PKC) in the carboxyl-terminal region and three characteristic antiparallel coiled coil (ACC) domain repeats in the amino-terminal region. The preferred peptide phosphorylation motif for PKNs determined by a combinatorial peptide library method was highly similar to that of PKCs within a 10 amino acid stretch. Previously reported PKN inhibitory compounds also inhibit PKCs to a similar extent, and no PKN selective inhibitors have been commercially available. We have identified a 15 amino acid peptide inhibitor of PKNs based on amino acids 485-499 of the carboxyl-terminal region of the C2-like domain of PKN1. This peptide, designated as PRL, selectively inhibi... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2926773 Mon, 26 Oct 2009 00:00:00 +0100 2926773 http://www.medworm.com/index.php?rid=2854944&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090908 Tumor necrosis factor-α (TNFα) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the nuclear factor κB (NFκB) signaling pathway particularly in macrophages has been implicated in many diseases. We demonstrate here that G-protein coupled receptor kinase-2 and 5 (GRK2 and 5) regulate TNFα-induced NFκB signaling in Raw264.7 macrophages. RNAi knockdown of GRK2 or 5 in macrophages significantly inhibits TNFα-induced IκBα phosphorylation and degradation, NFκB activation, and expression of the NFκB-regulated gene, macrophage inflammatory protein-1β. Consistent with these results, over-expression of GRK2 or 5 enhances TNFα-induced N... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2854944 Thu, 01 Oct 2009 23:00:00 +0100 2854944 http://www.medworm.com/index.php?rid=2795920&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091271 The protein kinase Tpl2 is activated by LPS, TNFα and IL-1. Activation of the native Tpl2 complex by these agonists requires the IKKβ-catalysed phosphorylation of the p105/NFκB1 subunit and is accompanied by the release of the catalytic subunit from both p105/NFκB1 and another subunit ABIN2. Here we report that IL-1 activates the transfected Tpl2 catalytic subunit in an HEK293 cell line that stably expresses the IL-1 receptor, but does not express the protein kinase IRAK1. In these cells IL-1 does not activate IKKβ or induce the phosphorylation of p105/NFκB1, and nor does the IKKβ inhibitor PS1145 prevent the IL-1-induced activation of transfected Tpl2. However, the IL-1-stimulated activation of transfected Tpl2 in IRAK1-null cells or acti... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2795920 Mon, 14 Sep 2009 23:00:00 +0100 2795920 http://www.medworm.com/index.php?rid=2792710&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090992 Integrin α5β1 is a key receptor for the extracellular matrix protein fibronectin.Antagonists of human α5β1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by α5β1 is poorly understood. Here we demonstrate that zebrafish α5β1 integrins do not interact with human fibronectin or the human α5β1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish α5β1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish α5 subunit β-propeller domain shows that these residues are important for the... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2792710 Sun, 13 Sep 2009 23:00:00 +0100 2792710 http://www.medworm.com/index.php?rid=2785391&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091144 This study reveals a new link between exercise and S-nitrosylation of skeletal muscle contractile proteins that may be important under (patho)physiological conditions. (Source: BJ Signal) BJ Signal journals http://www.medworm.com/rss/comments.php?id=2785391 Thu, 10 Sep 2009 23:00:00 +0100 2785391 http://www.medworm.com/index.php?rid=2785390&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090842 In adipocytes, Phosphodiesterase 3B (PDE3B) is an important regulatory effector in signaling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the β3-receptor agonist CL316243 (CL) indicated that insulin preferentially phosphorylated /activated PDE3B associated with internal membranes (endoplasmic reticulum/Golgi), whereas CL preferentially phosphorylated/ activated PDE3B associated with caveolae. siRNA-mediated knock-down (KD) of caveolin-1 (cav-1) in 3T3-L1 adipocytes resulted in down-regulation of expression of membrane- associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Similar results were obtained in adipocytes from cav-1 deficient mice. siRN... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2785390 Thu, 10 Sep 2009 23:00:00 +0100 2785390 http://www.medworm.com/index.php?rid=2751089&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082377 The Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Altered Rnd3 expression causes changes in cytoskeletal organization and cell cycle progression. Rnd3 functions to decrease RhoA activity, but how Rnd3 itself is regulated to cause these changes is still under investigation. Unlike other Rho family proteins, Rnd3 is regulated not by GTP/GDP cycling, but at the level of expression and by posttranslational modifications such as prenylation and phosphorylation. We show here that, upon PKC agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift and its subcellular localization becomes enriched at internal membranes. These changes are blocked by inhibition of conventional PKC isoforms and do not occur in PKCα-null cells or ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2751089 Mon, 31 Aug 2009 23:00:00 +0100 2751089 http://www.medworm.com/index.php?rid=2751088&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091170 Dietary isothiocyanates (ITCs) prevent cancer and show other bioactivities in vivo. As electrophiles, ITCs may covalently modify cellular proteins. Using a novel proteomics screen, we identified the Macrophage Migration Inhibitory Factor (MIF) as the principal target of nutrient ITCs in intact cells. ITCs covalently modify the amino-terminal proline residue of MIF and extinguish its catalytic tautomerase activity. MIF deficiency does not prevent induction of Phase 2 gene expression, a hallmark of many cancer chemopreventives including ITCs. Due to the emerging role of MIF in control of malignant cell growth and its clear involvement in inflammation, inhibition of MIF by nutrient ITCs suggests therapeutic strategies for inflammatory diseases and cancer. (Source: BJ Signal) BJ Signal journals http://www.medworm.com/rss/comments.php?id=2751088 Mon, 31 Aug 2009 23:00:00 +0100 2751088 http://www.medworm.com/index.php?rid=2743567&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090933 We have previously reported the design of an RI anchoring disruptor peptide (RIAD) that specifically displaces protein kinase A (PKA) type I from the A-kinase anchoring protein Ezrin, which is present in the immunological synapse of T cells. This increases immune reactivity by reducing the threshold for activation, and may prove a feasible approach for improving immune function in patients with cAMP-mediated T-cell dysfunction. However, the use of RIAD in biological systems is restricted by its susceptibility to enzymatic cleavage and, consequently, its short half-life in presence of the ubiquitous serum peptidases. In the present study, carefully selected non-natural amino acids were employed in the design of RIAD analogs with improved stability. The resulting peptidomimetics demonstrated... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2743567 Thu, 27 Aug 2009 23:00:00 +0100 2743567 http://www.medworm.com/index.php?rid=2740790&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090549 The metalloproteinases TACE (ADAM17) and ADAM10 are the primary enzymes responsible for catalyzing release of membrane anchored proteins from the cell surface in metazoan organisms. While the repertoire of protein substrates for these two proteases is partially overlapping, each one appears to target a subset of unique proteins in vivo. The mechanisms by which the two proteases achieve specificity for particular substrates are not completely understood. We have used peptide libraries to define the cleavage site selectivity of TACE and ADAM10. The two proteases have distinct primary sequence requirements at multiple positions surrounding the cleavage site in their substrates, which allowed us to generate peptide substrates that are highly specific for each of these proteases. The major diff... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2740790 Thu, 27 Aug 2009 23:00:00 +0100 2740790 http://www.medworm.com/index.php?rid=2719188&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090120 Hypoxia-inducible factor (HIF)-3alpha is the third member of the HIF transcription factor family. Whereas HIF-1alpha and -2alpha play critical roles in the cellular and systemic adaptation to hypoxia, little is known about the regulation and function of HIF-3alpha. At least five different splice variants may be expressed from the human HIF-3alpha locus, which are suggested to exert primarily negative regulatory effects on hypoxic gene induction. Here we report that hypoxia induces the human HIF-3alpha gene at the transcriptional level in a HIF-1-dependent manner. HIF-3alpha2 and HIF-3alpha4 transcripts, the HIF-3alpha splice variants expressed in Caki-1 renal carcinoma cells, rapidly increased after exposure to hypoxia or chemical hypoxia mimetics. SiRNA-mediated HIF-alpha knock-down demon... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2719188 Thu, 20 Aug 2009 23:00:00 +0100 2719188 http://www.medworm.com/index.php?rid=2715375&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090447 In this report, we provide evidence that the molecular machinery of uPAR-mediated VSMC differentiation employs lipid rafts. We show that the disruption of rafts in VSMC by membrane cholesterol depletion using methyl-β-cyclodextrin (MCD) or filipin leads to upregulation of uPAR and cell de-differentiation. uPAR silencing by means of interfering RNA resulted in an increased expression of contractile proteins. Consequently, disruption of lipid rafts impaired expression of these proteins and transcriptional activity of related genes. We provide evidence that this effect was mediated by uPAR. Similar effects were observed in VSMC isolated from caveolin-1 deficient mice. Despite the level of uPAR being significantly higher after the rafts’ disruption, uPA/uPAR-dependent cell migrat... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2715375 Wed, 19 Aug 2009 23:00:00 +0100 2715375 http://www.medworm.com/index.php?rid=2711560&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091010 Tie2 is a receptor tyrosine kinase expressed predominantly on the surface of endothelial cells. Activated by its ligands, the Angiopoietins, Tie2 initiates signaling pathways that modulate vascular stability and angiogenesis. Deletion of either Tie2 or Angiopoietin-1 (Ang1) genes in mice results in lethal vascular defects signifying their importance in vascular development. The mechanism employed by the Tie2 signalling machinery to attenuate or cause receptor trafficking is not well defined. Stimulation of Tie2-expressing cells with Ang1 results in its ubiquitylation suggesting that this may provide the necessary signal for receptor turnover. Using a candidate molecule approach, we demonstrate that Tie2 co-immunoprecipitates with c-Cbl in an Ang1-dependent manner and its ubiquitylation can... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2711560 Mon, 17 Aug 2009 23:00:00 +0100 2711560 http://www.medworm.com/index.php?rid=2711559&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090982 To date, 11 human cyclic nucleotide phosphodiesterase (PDE) families have been identified. Of these, five families contain non-catalytic tandem GAF domains (GAFa and GAFb) in the N-terminal part of the enzyme. For PDE2A, PDE5A and PDE6, the GAF domains have been shown to bind cyclic GMP with high affinity. For PDE2A and PDE5A, the ligand binding has been shown to stimulate the catalytic activity of the enzyme. For the most recently described PDEs, PDE10A and PDE11A, the GAF domains have previously been suggested to bind cAMP and cGMP, respectively. We have developed scintillation proximity based assays for cyclic nucleotide binding to PDE2A, PDE10A and PDE11A GAF domains. We directly demonstrate binding of cyclic nucleotides to the PDE10A and PDE11A GAF domains and show that these non-cata... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2711559 Mon, 17 Aug 2009 23:00:00 +0100 2711559 http://www.medworm.com/index.php?rid=2666764&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090854 Knowledge of the cellular targets of reactive oxygen species (ROS) and their regulation is an essential prerequisite for understanding ROS-mediated signaling. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is known as a major target protein in oxidative stresses and becomes thiolated in active site. However, the molecular and functional changes of oxidized GAPDH, inactive form, have not yet been characterized. To examine the modifications of GAPDH under oxidative stress, we separated the oxidation products by 2D-gel electrophoresis and identified them using nanoLC-ESI-q-TOF tandem MS. Intracellular GAPDH subjected to oxidative stress separated into multiple acidic spots on 2D-gel electrophoresis and were identified as cysteine disulfide and cysteic acids in active site C152. We identifi... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2666764 Mon, 03 Aug 2009 23:00:00 +0100 2666764 http://www.medworm.com/index.php?rid=2666765&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090228 The human family of MAP kinase signal-integrating kinases (Mnks) comprises four related proteins derived from two genes by alternative splicing. The Mnk1 gene gives rise to two proteins, Mnk1a and Mnk1b, which possess distinct C-termini and properties. Despite lacking the C-terminal MAP kinase-binding site, Mnk1b shows higher basal activity than Mnk1a. In contrast, the activity of Mnk1a is tightly regulated by signalling through ERK and p38 MAP kinase. We show that the short C-terminus of Mnk1b confers on it a ‘default’ behaviour of substantial, but unregulated, activity. In contrast, the longer C-terminus of Mnk1a represses the basal activity and T (activation)-loop phosphorylation of this isozyme while allowing both properties to be stimulated by upstream MAP kinase sig... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2666765 Sun, 02 Aug 2009 23:00:00 +0100 2666765 http://www.medworm.com/index.php?rid=2620460&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090366 The role of SGK1 (serum and glucocorticoid-inducible kinase 1) in the glucocorticoid-induced of α-ENaC (epithelial Na+ channel α subunit) gene transcription was explored by monitoring the transcriptional activity of a luciferase-linked, α-ENaC reporter gene construct (pGL3-KR1) expressed in H441 airway epithelial cells. Dexamethasone evoked a concentration-dependent (EC50 ~ 4 µM) increase in transcriptional activity dependent upon a glucocorticoid response element in the α-ENac sequence. Although dexamethasone also activated endogenous SGK1, artificially increasing cellular SGK1 activity by expressing a constitutively active SGK1 mutant (SGK1-S422D) in hormone-deprived cells did not activate pGL3-KR1. Moreover, expression of catalytically inactive ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2620460 Mon, 20 Jul 2009 23:00:00 +0100 2620460 http://www.medworm.com/index.php?rid=2620458&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090392 This study has investigated regulation of flagellin /TLR5 signalling in human colonocytes (HT29-19A) by interferon-gamma (IFNγ), a cytokine released early in the inflammatory process which has multiple effects on gut epithelial function that may facilitate abnormal responses to enteric bacteria. Flagellin induced a dose-dependent secretion of chemokines CXCL8 and CCL2 in the human colonocyte line, HT29-19A. Exposure to IFNγ did not induce chemokine secretion but markedly potentiated responses to flagellin increasing CXL8 gene expression and protein secretion by ~4 fold. Potentiation by IFNγ was independent of changes in TLR5 and associated with a rapid, sustained increase in expression of the downstream adaptor molecule MyD88. Knockdown of MyD88 expression using siRNA ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2620458 Mon, 20 Jul 2009 23:00:00 +0100 2620458 http://www.medworm.com/index.php?rid=2613395&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090241 In this study we use Saccharomyces cerevisiae mutants to examine the potential roles of inositol pyrophosphates in responding to cell damage caused by reactive oxygen species (ROS). Yeast lacking kcs1 (the S. cerevisiae IP6K) have greatly reduced IP7 and IP8 levels, and display increased resistance to cell death caused by hydrogen peroxide (H2O2) consistent with a sustained activation of DNA repair mechanisms controlled by the Rad53 pathway. Other Rad53 controlled functions such actin polymerization appear unaffected by inositol pyrophosphates. Yeast lacking vip1 (S. cerevisiae PP-IP5K) accumulate large amounts of the inositol pyrophosphate IP7, but have no detectable IP8, indicating that this enzyme represents the physiological IP7-Kinase. Similar to kcs1Δ yeast, vip1Δ cells... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2613395 Thu, 16 Jul 2009 23:00:00 +0100 2613395 http://www.medworm.com/index.php?rid=2605361&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090687 Phosphoinositide 3-kinase (PI3K) has been implicated in phagocytosis and fluid-phase pinocytosis in macrophages. The subtype-specific role of PI3K in these processes is poorly understood. To elucidate this issue, we made Raw 264.7 cells deficient in each of the class-I PI3K catalytic subunits: p110α, p110β, p110δ and p110γ. Among these cells, only the p110α-deficient cells exhibited lower phagocytosis of opsonized and non-opsonized zymosan. The p110α-deficient cells also showed the impaired phagocytosis of IgG-opsonized erythrocytes and the impaired fluid-phase pinocytosis of dextran (MW 40,000). Receptor-mediated pinocytosis of DiI-labeled acetylated LDL and fluid-phase pinocytosis of lucifer yellow (MW 500) were resistant to p110α depletio... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2605361 Tue, 14 Jul 2009 23:00:00 +0100 2605361 http://www.medworm.com/index.php?rid=2597589&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090208 Interleukin-1 (IL-1) is a pro-inflammatory cytokine that has a variety of effects during the process of inflammation. Stimulating cells with IL-1 initiates a signaling cascade, which includes the activation of the nuclear transcription factor-κB (NF-κB), and subsequently induces a variety of inflammatory genes. Although the molecular mechanism for the IL-1-induced activation of NF-κB has been well documented, much less is known about the mechanism by which protein phosphatases downregulate this pathway. Here we show that mouse protein phosphatase 2Cη-2 (PP2Cη-2), a novel member of the protein serine/threonine phosphatase 2C family, inhibits the IL-1-NF-κB signaling pathway. Ectopic expression of PP2Cη-2 in HEK293IL-1RI cells inhibited the IL... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2597589 Mon, 13 Jul 2009 23:00:00 +0100 2597589 http://www.medworm.com/index.php?rid=2569156&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090107 Mitochondrial thioredoxin (Trx2) is an antioxidant, antiapoptotic factor, essential for cell viability. The cytoplasmic thioredoxin (Trx1) is a cofactor and regulator of redox sensitive transcription factors such as the glucocorticoid receptor (GR) and NF-κB. Both transcription factors have been detected in mitochondria and a role in mitochondrial transcription regulation and apoptosis has been proposed. Here, we show using surface plasmon resonance and immunoprecepitation that GR and the p65 subunit of NF-κΒ are Trx2 interacting proteins. The interaction of Trx2 with GR is independent of the presence of GR ligand and of redox conditions. The p65 subunit of NF-κB can interact with Trx2 in the oxidized, but not the reduced form. Using HEK 293 cell lines with incr... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2569156 Wed, 01 Jul 2009 23:00:00 +0100 2569156 http://www.medworm.com/index.php?rid=2521659&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090342 Transcriptional regulation of the p53 tumor suppressor gene plays an important role in expression control of various target genes involved in the DNA damage response. However, the molecular basis for this regulation remains obscure. Here we demonstrate that RAS-responsive element-binding protein-1 (RREB-1) efficiently binds to the p53 promoter via the p53 core promoter element and transactivates p53 expression. Silencing of RREB-1 significantly reduces p53 expression at both the mRNA and the protein levels. Notably, disruption of RREB-1-mediated p53 transcription suppresses the expression of the p53 target genes. We also show that, upon exposure to genotoxic stress, RREB-1 controls apoptosis in a p53-dependent manner. These findings provide evidence that RREB-1 participates in modulating p... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2521659 Thu, 25 Jun 2009 23:00:00 +0100 2521659 http://www.medworm.com/index.php?rid=2516816&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090735 Hypertension secondary to scavenging of nitric oxide (NO) remains a limitation in the use hemoglobin based oxygen carriers (HBOCs). Recent studies suggest that nitrite reduction to NO by deoxyhemoglobin supports NO-signaling. Herein, we tested whether nitrite would attenuate HBOC-mediated hypertension using HBOC-201 (Biopure), a bovine cross-linked, low oxygen affinity hemoglobin. Similar to unmodified hemoglobin, deoxygenated HBOC-201 reduced nitrite to NO with rates directly proportional to the extent of deoxygenation. The functional importance of HBOC-201 dependent nitrite reduction was demonstrated using isolated aortic rings and a murine model of trauma, hemorrhage and resuscitation. In the former, HBOC-201 inhibited NO-donor and nitrite-dependent vasodilation when oxygenated. However... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2516816 Wed, 24 Jun 2009 23:00:00 +0100 2516816 http://www.medworm.com/index.php?rid=2503393&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090194 NAADP is a novel second messenger thought to mobilize acidic Ca2+ stores that are functionally coupled to the endoplasmic reticulum. Although NAADP-sensitive Ca2+ stores have been described in neurons, the physiological cues that recruit them are not known. Here we show that in both hippocampal neurons and glia, extracellular application of glutamate in the absence of external Ca2+ evoked cytosolic Ca2+ signals that were inhibited by blockade of V-type ATPases or following osmotic bursting of lysosomes. The sensitivity of both cell types to glutamate correlated well with lysosomal Ca2+ content. However, interfering with acidic compartments was largely without effect on the Ca2+ content of the endoplasmic reticulum or Ca2+ signals in response... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2503393 Mon, 22 Jun 2009 23:00:00 +0100 2503393 http://www.medworm.com/index.php?rid=2503392&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090638 The generation of various phosphoinositide messenger molecules at distinct locations within the cell is mediated via the specific targeting of different isoforms and splice variants of phosphoinositide kinases. The lipid messenger phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is generated by several of these enzymes when targeted to distinct cellular compartments. Several splice variants of the type Iγ isoform of phosphatidylinositol 4-phosphate, 5-kinase (PIPKIγ) which generate PtdIns(4,5)P2 have been identified, and each splice variant is thought to serve a unique functional role within cells. Here, we have identified two novel C-terminal splice variants of PIPKIγ in human cells consisting of 700 and 707 amino acids. These two splice variants are expressed in... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2503392 Mon, 22 Jun 2009 23:00:00 +0100 2503392 http://www.medworm.com/index.php?rid=2503395&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090339 Nitro-fatty acid products of oxidative inflammatory reactions mediate anti-inflammatory cell signaling responses. Nitro-linoleic acid (LNO2) induces expression of heme oxygenase-1 (HO-1), an enzyme that catabolizes heme into products exhibiting potent anti-inflammatory properties. Here the molecular mechanisms underlying HO-1 induction by LNO2 were examined in human aortic endothelial cells (HAEC), human embryonic kidney (HEK) 293 cells, and in transcription factor-deficient mouse embryonic fibroblasts (MEF). LNO2 induced HO-1 expression in Nrf2 deficient MEF and in HEK 293 cells transfected with Nrf2 specific shRNA, supporting that LNO2-mediated HO-1 induction can be regulated by Nrf2-independent mechanisms. LNO2 activated expression of a -4.5 kb human HO-1 promoter construct, while a -4.... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2503395 Wed, 17 Jun 2009 23:00:00 +0100 2503395 http://www.medworm.com/index.php?rid=2503394&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090055 The molecular complex containing BCL10 and CARMA proteins has been recently identified as key component in the signalling transduction pathways that regulate activation of NF-κB transcription factor in lymphoid and non-lymphoid cells. Assembly of the molecular complexes containing BCL10 and CARMA proteins relies on homophylic interactions established between the CARD domains of these proteins. In order to identify BCL10 inhibitory peptides, we have established a method of assaying peptides derived from the CARD of BCL10 in a binding competition assays of CARD-CARD self-association. By this procedure, a short peptide corresponding to the amino acidic residues 91-98 of BCL10 has been selected as an effective inhibitor of the protein self-association. When tested in cell assays for its... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2503394 Wed, 17 Jun 2009 23:00:00 +0100 2503394 http://www.medworm.com/index.php?rid=2503396&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082182 Rac1 and Rac2, which belong to the Rho subfamily of Ras-related GTPases, play an essential role in activation of gp91phox (also known as Nox2), the catalytic core of the superoxide-producing NADPH oxidase in phagocytes; and Rac1 also contributes to activation of the non-phagocytic oxidases Nox1 and Nox3, each related closely to gp91phox/Nox2. It has remained controversial whether the insert region of Rac (amino acids 123–135), unique to the Rho subfamily proteins, is involved in gp91phox/Nox2 activation. Here we show that removal of the insert region from Rac1 neither affects activation of gp91phox/Nox2, which is reconstituted under cell-free and whole-cell conditions, nor blocks its localization to phagosomes during ingestion of IgG-coated beads by macrophage-like RAW264.7 cells. T... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2503396 Tue, 16 Jun 2009 23:00:00 +0100 2503396 http://www.medworm.com/index.php?rid=2503397&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090764 The mechanisms involved in sensing oxidative signaling molecules such as H2O2 in plant and animal cells are not completely understood. In the present study, we tested the postulate that oxidation of methionine (Met) to Met sulfoxide (MetSO) can couple oxidative signals to changes in protein phosphorylation. We demonstrate that when a Met residue functions as a hydrophobic recognition element within a phosphorylation motif, its oxidation can strongly inhibit peptide phosphorylation in vitro. This is shown to occur with recombinant soybean calcium dependent protein kinases (CDPKs) and human AMP-dependent protein kinase (AMPK). To determine whether this effect may occur in vivo, we monitored the phosphorylation status of Arabidopsis leaf nitrate reductase (NR) Ser534 using modification-specif... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2503397 Mon, 15 Jun 2009 23:00:00 +0100 2503397 http://www.medworm.com/index.php?rid=2467842&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090428 We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesised by different PI 4-kinase activities, and also the presence of PtdIns4P in cytoplasmic vesicles which are not readily identifiable as PI 4-kinase containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P2. (Source: BJ Signal) BJ Signal journals http://www.medworm.com/rss/comments.php?id=2467842 Tue, 09 Jun 2009 04:00:00 +0100 2467842 http://www.medworm.com/index.php?rid=2455840&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090654 In this study we test the hypothesis that PI3K-C2β translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2β resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme in the cytosol and then in nuclei. Using detergent treated nuclei, PI3K-C2β co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically and phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2β levels and lipid kinase activity following epidermal growth factor stimulation. C-terminal deletion and point mutation of PI3K-C2β demonstrated that epidermal growth factor driven translocation to... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2455840 Thu, 04 Jun 2009 04:00:00 +0100 2455840 http://www.medworm.com/index.php?rid=2455841&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082308 Cyclic ADP-ribose (cADPR) is a universal Ca2+ mobilizing second messenger. In T cells cADPR is involved in sustained Ca2+ release and also in Ca2+ entry. Potential mechanisms for the latter include either capacitative Ca2+ entry secondary to store depletion by cADPR or direct activation of the non-selective cation channel transient receptor potential – melastatin type 2 (TRPM2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-bromo-cyclic inosine diphosphoribose (8-Br-N1-cIDPR). 8-Br-N1-cIDPR evoked Ca2+ signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca2+ signalling induced by 8-Br-N1-cIDPR consisted of Ca2+ release and Ca2+ entry. While ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2455841 Wed, 03 Jun 2009 04:00:00 +0100 2455841 http://www.medworm.com/index.php?rid=2455842&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090471 Nrf2 is a transcription factor that activates transcription of a battery of cytoprotective genes by binding to the antioxidant response element (ARE). Nrf2 is repressed by the cysteine-rich Keap1 protein, which targets Nrf2 for ubiquitination and subsequent degradation by a Cul3-mediated ubiquitination complex. We find that modification of C151 of human Keap1 by mutation to a tryptophan relieves the repression by Keap1 and allows activation of the ARE by Nrf2. Keap1 C151W has a decreased affinity for Cul3, and can no longer serve to target Nrf2 for ubiquitination, though it retains its affinity for Nrf2. A series of 12 mutant Keap1 proteins, each containing a different residue at position 151, was constructed to explore the chemistry required for the effect. The series reveals that the ext... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2455842 Tue, 02 Jun 2009 04:00:00 +0100 2455842 http://www.medworm.com/index.php?rid=2455843&cid=s_37620_60_f&fid=37620&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090341 In this study, we isolated the full-length cDNA and studied the role of human SCUBE2 in the HH signaling cascade. When overexpressed, recombinant human SCUBE2 manifests as a secreted, surface-anchored glycoprotein. Deletion mapping analysis defines the critical role of the spacer region and/or cystein-rich repeats for membrane association. Further biochemical analyses and functional reporter assays demonstrated that human SCUBE2 can specifically interact with Sonic HH (SHH) and SHH receptor Patched-1 (PTCH1), and enhance the SHH signaling activity within the cholesterol-rich raft microdomains of the plasma membranes. Together, our results reveal that human SCUBE2 is a novel, positive component of the HH signal, acting upstream in ligand reception at the plasma membrane. Thus, human SCUBE2 ... BJ Signal journals http://www.medworm.com/rss/comments.php?id=2455843 Mon, 01 Jun 2009 04:00:00 +0100 2455843