Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent studies of the FGR protooncogene have shown that expression of its mRNA is limited to mature peripheral blood granulocytes, monocytes, and tissue macrophages. In the present study, we have investigated p55c-fgr expression in normal human neutrophils [polymorphonuclear leukocytes (PMN)] and have found enzymatically active p55c-fgr to be abundant in lysates of PMN and murine fibroblasts transfected with a FGR expression plasmid but not control cells. Fractionation studies revealed that neutrophil p55c-fgr was present in plasma membrane-enriched fractions as well as fractions containing secondary and tertiary granules. Little change in the distribution of p55c-fgr or FGR kinase activity was observed under conditions favoring tertiary granule release. In contrast, when secondary granule secretion was induced with the chemoattractant peptide, formyl-Met-Leu-Phe, a marked decrease in p55c-fgr and FGR kinase was observed in fractions depleted of secondary granules. Concomitantly, the relative concentration of p55c-fgr and its enzymatic activity were increased in fractions containing plasma membrane. From these findings we conclude that p55c-fgr is associated with functional secretory granules and is redistributed within normal neutrophils in response to their activation.
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PMID:Translocation of the FGR protein-tyrosine kinase as a consequence of neutrophil activation. 268 59

We have examined the sites phosphorylated on acetyl-CoA carboxylase by three protein kinases which have been shown to inactivate the enzyme, i.e. cyclic-AMP-dependent protein kinase, acetyl-CoA carboxylase kinase-2 (ACK2, purified from rat mammary gland) and the AMP-activated protein kinase (formerly called acetyl-CoA carboxylase kinase-3, purified from rat liver). Each protein kinase phosphorylates two out of three sites (termed 1-3) which have been established by amino acid sequencing. The two sites phosphorylated by each kinase can be recovered on separate peptides, TC1 and TC2, derived by combined digestion of the native enzyme by trypsin and chymotrypsin: TC1 = Ser-2Ser(P)-Met-3Ser(P)-Gly-Leu; TC2 = Arg-Met-1Ser(P)-Phe- Cyclic-AMP-dependent protein kinase phosphorylates sites 1 and 2 exclusively, whereas the AMP-activated protein kinase phosphorylates sites 1 and 3, plus at least one other minor site. ACK2 phosphorylates site 1 and, more slowly, an unidentified site(s) within TC1. We have also established the structures of the single major phosphopeptides (T1 and C1 respectively) which are recovered by HPLC after acetyl-CoA carboxylase phosphorylated by cyclic-AMP-dependent protein kinase is digested with trypsin or chymotrypsin alone. T1 is related to TC1, and has the structure: Ser-Ser(P)-Met-Ser-Gly-Leu-His-Leu-Val-Lys. C1 is identical with TC2. We have carried out studies on the correlation of the activity of acetyl-CoA carboxylase with the occupancy of sites 1, 2 and 3 during phosphorylation by each of the three protein kinases. The results suggest that phosphorylation of site 3 is primarily responsible for the large decrease in Vmax produced by the AMP-activated protein kinase, while phosphorylation of site 1 may be primarily responsible for the increase in A0.5 for citrate and more modest depression of Vmax produced by cyclic-AMP-dependent protein kinase and ACK2. Our results emphasize that amino acid sequence information is essential in the unequivocal interpretation of data from phosphopeptide mapping experiments and allow a more complete interpretation of previous data on phosphorylation of acetyl-CoA carboxylase in intact cells. They also open the way to experiments which could establish the physiological roles of these protein kinases in the control of fatty acid synthesis.
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PMID:Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-CoA carboxylase. 290 Jan 38

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.
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PMID:p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation. 756 38

The clinical isolate Escherichia coli PEY was highly resistant to amoxycillin, ticarcillin and piperacillin associated to beta-lactamase inhibitors such as clavulanic acid, sulbactam, tazobactam and brobactam but susceptible to cephalosporins, aztreonam and imipenem. The susceptibility to mecillinam indicated that this phenotype was not related to hyperproduction of the TEM-1 beta-lactamase. E. coli PEY produced a new plasmid-mediated inhibitor-resistant beta-lactamase of pI 5.2, which was named IRT-4. The determination of the amino acid sequence (Swiss-Prot accession number, P00810) of the purified protein indicated that IRT-4 differed from TEM-1 by two substitutions: Leu for Met-69 (ABL numbering) and Asp for Asn-276. A Met-69-Leu variant of TEM-1, obtained by site-directed mutagenesis, has been described as resistant to clavulanate. The Asp for Asn-276 substitution has not been reported previously. The side chains of Asp-276 and Arg-244 were expected to interact. Determinations of 50% inhibitory concentrations of beta-lactamase inhibitors and substrate profile of IRT-4 suggested that such an ionic bond was implicated in the alteration of the mechanistic process of TEM-1 beta-lactamase.
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PMID:Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to beta-lactamase inhibitors. 805 82

The absence of CTLA-4 results in uncontrolled T cell proliferation. The T cell receptor-specific kinases FYN, LCK, and ZAP-70 as well as the RAS pathway were found to be activated in T cells of Ctla-4-/- mutant mice. In addition, CTLA-4 specifically associated with the tyrosine phosphatase SYP, an interaction mediated by the SRC homology 2 (SH2) domains of SYP and the phosphotyrosine sequence Tyr-Val-Lys-Met within the CTLA-4 cytoplasmic tail. The CTLA-4-associated SYP had phosphatase activity toward the RAS regulator p52SHC. Thus, the RAS pathway and T cell activation through the T cell receptor are regulated by CTLA-4-associated SYP.
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PMID:Regulation of T cell receptor signaling by tyrosine phosphatase SYP association with CTLA-4. 863 61

Constitutive activation of growth factor receptors through autocrine/paracrine mechanisms occurs frequently in human cancers and is thought to play an important role in carcinogenesis. We have demonstrated previously that hepatocyte growth factor (HGF) is a potent mitogenic factor for murine mammary carcinoma (SP1) cells in vitro. We report here an autocrine HGF loop in SP1 cells. HGF receptor/Met is expressed in SP1 cells and is constitutively tyrosine phosphorylated. The phosphorylation of HGF receptor/Met is inhibited when cells are exposed to suramin or anti-HGF IgG. This finding suggests that constitutive tyrosine phosphorylation of HGF receptor/Met is sustained by an extracellular factor, most likely HGF. Using Northern blot and Western blot analysis, we detected expression of a 6-kb HGF mRNA in SP1 cells and a M(r) 85,000 HGF protein in SP1-conditioned medium, respectively. In vitro translation of mRNA from SP1 cells and metabolic labeling confirmed expression and synthesis of HGF by SP1 cells. SP1 cells also invade through Matrigel-coated transwell membranes in an in vitro invasion assay, and invasion of these cells was inhibited by neutralizing anti-HGF IgG. In addition, SP1-conditioned medium induced scatter activity of Madin-Darby canine kidney epithelial cells, and this activity was inhibited by neutralizing anti-HGF IgG. We have also shown that several signaling molecules including phosphatidylinositol 3-kinase, Src, focal adhesion kinase, and phospholipase C-gamma in SP1 cells are constitutively tyrosine phosphorylated, suggesting that coexpression of HGF and HGF receptor/Met may in part contribute to sustained tyrosine phosphorylation of these cytoplasmic proteins in SP1 cells. Our observations in the SP1 model suggest that HGF contributes to growth and invasive phenotypes of mammary carcinomas via both paracrine and autocrine mechanisms.
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PMID:Identification of a hepatocyte growth factor autocrine loop in a murine mammary carcinoma. 882 10

To investigate the in vivo role of hepatocyte growth factor/scatter factor (HGF/SF) in liver function, we generated transgenic mice using a mouse HGF/SF cDNA under the control of the mouse metallothionein gene promoter and 5'/3' flanking sequences. In adult HGF/SF transgenic mice, liver weight as a percentage of total body weight was at least twice that of wild-type mice. Comparison of transgenic and control liver morphology revealed dramatic heterogeneity in the size and appearance of hepatocytes as a distinctive feature of HGF/SF overexpression. Transgenic livers exhibited a significant increase in the number of small hepatocytes with a 2N DNA content, accounting for the observed increase in liver mass. The DNA labeling index of hepatocytes increased 11-fold at 4 weeks of age, when liver enlargement first became apparent, and was still elevated about 5-fold in adult HGF/SF transgenic mice. Moreover, hepatocytes isolated by perfusion of transgenic livers doubled every 2 days in culture, whereas little or no growth was observed with isolated control hepatocytes. The mechanistic basis of hepatocyte proliferation was elucidated as the chronic activation of the c-met proto-oncogene product. Met and substrates such as phosphatidylinositol 3-kinase, Src homology and collagen-like, pp60c-src, focal adhesion kinase p125FAK, and paxillin were associated with tyrosine-phosphorylated complexes in a hepatocyte cell line established from the transgenic liver. This proliferative stimulus triggered the formation of hepatocellular adenomas and/or carcinomas in most transgenic mice > or = 1.5 years of age. Finally, the rate of transgenic mouse liver regeneration was increased 3-fold over control livers following partial hepatectomy.
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PMID:Hepatocyte growth factor/scatter factor overexpression induces growth, abnormal development, and tumor formation in transgenic mouse livers. 893 Apr 1

Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfation of DHEA and other hydroxysteroids. DHEA ST enzymatic activity in individual human liver biopsy samples has been shown to vary over a five-fold range, and frequency distribution histograms are bimodal, with approximately 25% of subjects included in a high activity subgroup. We set out to characterize the molecular basis for variation in human liver DHEA ST activity. The first step involved performing quantitative Western analysis of cytosol preparations from 92 human liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. There was a highly significant correlation (r(s) = 0.635, P < 0.0001) between levels of DHEA ST activity and immunoreactive protein. We next attempted to determine whether the expression of DHEA ST might be controlled, in part, by a genetic polymorphism. DNA was isolated from three "low" and three "high" DHEA ST activity liver samples. Exons and the 5'-flanking region of the DHEA ST gene (STD) were amplified for each of these samples with the polymerase chain reaction (PCR). When compared with "wild type" STD sequence, some of the samples contained a T --> C transition at DHEA ST cDNA nucleotide 170, located within exon 2, resulting in a Met 57 --> Thr change in amino acid. Other samples contained an A --> T transversion at nucleotide 557 within STD exon 4 that resulted in a Glu 186 --> Val change. STD exons 2 and 4 were then sequenced for DNA isolated from an additional 87 liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. The allele frequency for the exon 2 polymorphism in these samples was 0.027, whereas that for the exon 4 polymorphism was 0.038, but neither polymorphism was systematically related to the level of enzyme activity in these samples. Transient expression in COS-1 cells of cDNA that contained the nucleotide 170 and 557 polymorphisms, either separately or together, resulted in decreased expression of both DHEA ST enzymatic activity and level of immunoreactive protein, but only when the nucleotide 557 variant was present. Identification of common genetic polymorphisms within STD will now make it possible to test the hypothesis that those polymorphisms might alter in vivo expression and/or function of this important human steroid-metabolizing enzyme.
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PMID:Human dehydroepiandrosterone sulfotransferase pharmacogenetics: quantitative Western analysis and gene sequence polymorphisms. 901 Mar 52

We have investigated the role of the glycine recognition site of the N-methyl-D-aspartate receptor (the GlyNMDA site) in the facilitation of NMDA receptor agonist-evoked activity in rat dorsal horn neurons that is brought about by neurokinin1 (NK1) receptor agonist and the contribution of protein kinase C (PKC) activation to this phenomenon. Ionophoresis of the selective NMDA receptor agonist 1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACBD) produced a sustained increase in the firing rate of single laminae III-V neurons recorded extracellularly using multibarrelled glass electrodes. The highly selective NK1 receptor agonist acetyl-[Arg6,Sar9,Met(O2)11]-SP6-11 (Sar9-SP) greatly facilitated this response, but under the present conditions had no effect when applied alone or with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor agonist) at the same current. In the presence of the GLyNMDA site antagonists 2-carboxy-4,6-dichloro-(1H)-indole-3-propanoic acid (MDL 29951), 7-chloro-3-(cyclopropylcarbonyl)-4-hydroxy-2(1H)-quinoline (L701,252), 5,7-dinitroquinaxoline-2,3-dione (MNQX) or 7-chlorothiokynurenic acid (7-CTK), or the PKC inhibitors, chelerythrine or GF109203X, the Sar9-SP-induced facilitation of ACBD-evoked activity was prevented, generally restoring activity to a level similar to that in the presence of ACBD alone, whilst an AMPA receptor antagonist, 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX) did not inhibit the facilitation. At the same ionophoretic currents these compounds had no effect on ACBD-evoked activity in the absence of Sar9-SP but were inhibitory at significantly greater currents. To further substantiate the importance of the GlyNMDA site in the interaction, the effects of NMDA receptor antagonists selective for alternative recognition sites on the NMDA receptor were investigated. MK-801, a non-competitive NMDA receptor antagonist and arcaine, a competitive inhibitor at the polyamine site, were applied to the facilitated activity seen in the presence of Sar9-SP and ACBD, and to ACBD-evoked activity alone. Unlike the GlyNMDA site antagonists and PKC inhibitors, these compounds reduced both facilitated and ACBD-evoked activity at similar currents. Furthermore, like the NK1 receptor agonist, a selective GlyNMDA site agonist 1-aminocyclopropane carboxylic acid (ACPC) caused facilitation of ACBD-evoked activity which was also blocked by currents of L701,252 that did not alter activity evoked by ACBD alone. These data suggest that activation of the GlyNMDA site (perhaps as a consequence of glycine release or modification of its influence by intracellular signalling cascades) is an essential component of the means by which NK1 receptor activation results in facilitated responsiveness of dorsal horn neurons towards NMDA receptor agonists.
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PMID:The glycine site of the NMDA receptor contributes to neurokinin1 receptor agonist facilitation of NMDA receptor agonist-evoked activity in rat dorsal horn neurons. 902 83

Vascular smooth muscle cell (VSMC) proliferation still remains a poorly understood process, although it is believed to play a critical role in pathological states, including atherosclerosis and hypertension. Several reports have suggested that proteases may be directly involved in this process; however, it was still unclear which protease is responsible for VSMC proliferation. In this study, by use of a cell-permeable calpain inhibitor (calpeptin; benzyloxycarbonyl-Leu-nLeu-H), its analogue (benzyloxycarbonyl-Leu-Met-H), the cell-impermeable serine protease inhibitor leupeptin, and antisense oligonucleotide against m-calpain to inhibit proliferation of primarily cultured human VSMCs, we investigated whether calcium-activated neutral protease (calpain) is involved in VSMC proliferation. Calpeptin and its analogue, more specific for m-calpain, equally inhibited the proliferation of VSMCs in a dose-related manner, whereas a more limited antiproliferative effect was observed in leupeptin-treated VSMCs. Antisense oligonucleotide against m-calpain, but not scrambled antisense, dose-dependently inhibited m-calpain expression and proliferation of VSMCs. Maximal inhibition was an approximately 50% reduction of cell number and m-calpain antigen observed at 50 micromol/L of antisense oligonucleotide. Calpeptin or antisense oligonucleotide against m-calpain increased the expression of the endogenous calpain substrate pp125FAK (focal adhesion kinase), whereas the expression of the endogenous calpain inhibitor calpastatin was not affected. These results suggest that the proliferation of VSMCs requires protease activity, some of which is due to m-calpain.
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PMID:Possible involvement of m-calpain in vascular smooth muscle cell proliferation. 951 20


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