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)

Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins such as cefotaxime and ceftazidime. To identify other amino acid substitutions that alter the activity of TEM-1 toward extended-spectrum cephalosporins, a random library was constructed that contained all possible amino acid substitutions over the 3-residue window of 238-241 (ABL numbering). Mutants were selected for 100-fold greater ceftazidime resistance than wild-type. All mutants had a serine substitution at position 238, a lysine or arginine at position 240, and a small amino acid at position 241. The role of each substitution was investigated by constructing individual G238S, E240K, and R241G substitutions as well as the G238S:E240K double mutant. Each enzyme was purified to homogeneity and the kinetic parameters kcat and Km were determined using several substrates. The G238S substitution increases catalytic efficiency for both ceftazidime and cefotaxime. However, to achieve large increases in catalytic efficiency, both G238S and the E240K substitutions are required. The R241G substitution results in a small increase in catalytic efficiency for only ceftazidime. The contribution of each residue to the transition-state stabilization energy was found to be additive indicating that the substitutions act independently to change the catalytic properties of the enzyme.
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PMID:Characterization of TEM-1 beta-lactamase mutants from positions 238 to 241 with increased catalytic efficiency for ceftazidime. 808 10

The tyrosine protein kinase p56lck transduces signals important for antigen-induced T-cell activation. In transgenic mice, p56lck is oncogenic when overexpressed or expressed as a mutant, catalytically activated enzyme. In humans, the LCK gene is located at the breakpoint of the t(1;7)(p34;q34) chromosomal translocation. This translocation positions the beta T-cell receptor constant region enhancer upstream of the LCK gene without interrupting the LCK coding sequences, and a translocation of this sort occurs in both the HSB2 and the SUP-T-12 T-cell lines. We have found that, although the level of the p56lck protein in HSB2 cells is elevated approximately 2-fold in comparison with that in normal T-cell lines, total cellular tyrosine protein phosphorylation is elevated approximately 10-fold. Increased levels of phosphotyrosine in HSB2 cells resulted from mutations in the LCK gene that activated its function as a phosphotransferase and converted it into a dominant transforming oncogene. The oncogenic p56lck in HSB2 cells contained one amino acid substitution within the CD4/CD8-binding domain, two substitutions in the kinase domain, and an insertion of Gln-Lys-Pro (QKP) between the SH2 and kinase domains. In NIH 3T3 fibroblasts, three of these mutations cooperated to produce the fully oncogenic form of this p56lck variant. These results suggest that mutation of LCK may contribute to some human T-cell leukemias.
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PMID:Oncogenic activation of the Lck protein accompanies translocation of the LCK gene in the human HSB2 T-cell leukemia. 813 46

To elucidate the role of cytokine receptor signal transduction in T-cell development, we have investigated the expression pattern and biochemical characteristics of the murine Janus family tyrosine kinase, JAK3. Previous studies have shown that JAK3 is expressed in lymphoid and myeloid tumor cell lines and in a small number of lymphoid tissues. To further characterize JAK3 expression, we used a quantitative polymerase chain reaction approach to compare JAK3 mRNA levels at multiple stages of T-cell differentiation and in a broad range of mouse tissues. These studies, in conjunction with analyses of JAK3 protein expression, show that the highest levels of JAK3 are in adult, 2-week-old, and fetal thymus, followed by somewhat lower levels in bone marrow, spleen, fetal liver, and adult CD4-CD8- thymocytes. We also show that different forms of JAK3 mRNA arise by alternative splicing. Finally, our biochemical studies show that the JAK3 kinase domain, but not the pseudo-kinase domain, has tyrosine kinase activity and, furthermore, that JAK3 kinase activity is abolished by an amino acid substitution of the conserved lysine in the kinase domain (K851R). These studies show that JAK3 expression is profoundly skewed to hematopoietic and lymphoid precursor cells, strongly suggesting a role for JAK3 in hematopoiesis and T- and B-cell development.
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PMID:Murine JAK3 is preferentially expressed in hematopoietic tissues and lymphocyte precursor cells. 860 29

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

Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton-insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells.
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PMID:Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein. 864 1

Tyk2 and JAK1, members of the Janus kinase (JAK) family of protein tyrosine kinases, are required for interferon-alpha/beta binding and signaling. Both enzymes are associated with the interferon-alpha/beta receptor, and upon ligand binding, they undergo tyrosine phosphorylation and catalytic activation in an interdependent manner. To identify residues involved in Tyk2 regulation and to understand the basis of the interdependence of Tyk2 and JAK1, six mutated versions of Tyk2 bearing single or multiple point mutations in the tyrosine kinase domain were studied in a cell line lacking endogenous Tyk2. The Y1054F/Y1055F substitutions in the putative activation loop prevented ligand-dependent activation of Tyk2, without abolishing its catalytic potential. The K930R mutation in the ATP binding site generated a kinase-negative protein, which however, still became phosphorylated upon interferon-alpha treatment. The Y1054F/Y1055F substitutions in this kinase-negative Tyk2 abolished the induced phosphorylation. These results indicate that Tyk2 is activated by phosphorylation on Tyr-1054 and/or Tyr-1055 and that this phosphorylation requires another kinase, most likely JAK1. While the Tyk2 forms mutated on Tyr-1054 and Tyr-1055 or on Lys-930 allowed some inducible gene expression, the combination of the three point mutations totally abolished signaling.
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PMID:Interferon-alpha-dependent activation of Tyk2 requires phosphorylation of positive regulatory tyrosines by another kinase. 870 90

Recently, natural variants of TEM-1 beta-lactamase with amino acid substitutions at residues 237-240 have been identified that have increased hydrolytic activity for extended-spectrum antibiotics such as ceftazidime. To identify the sequence requirements in this region for a given antibiotic, a random library was constructed that contained all possible amino acid combinations for the 3-residue region 237-240 (ABL numbering system) of TEM-1 beta-lactamase. An antibiotic disc diffusion method was used to select mutants with wild-type level activity or greater for the extended-spectrum cephalosporin ceftazidime and the monobactam aztreonam. Mutants that were selected for optimal ceftazidime hydrolysis contained a conserved Ala at position 237, a Ser for Gly substitution at position 238, and a Lys for Glu at position 240. Mutants selected for aztreonam hydrolysis exhibited a Gly for Ala substitution at position 237, a Ser for Gly substitution at position 238, and a Lys/Arg for Glu at position 240. The role of the A237G substitution in differentiating between ceftazidime and aztreonam was further investigated by kinetic analysis of the A237G, E240K, G238S:E240K, and A237G:G238S:E240K enzymes. The A237G single mutant and the G238S:E240K double mutant exhibited increases in catalytic efficiency for both ceftazidime and aztreonam. However, the triple mutant A237G:G238S:E240K, displayed a 12-fold decrease in catalytic efficiency for ceftazidime but a 3-fold increase for aztreonam relative to the G238S:E240K double mutant. Thus, the A237G substitution increases ceftazidime hydrolysis when present alone but antagonizes ceftazidime hydrolysis when it is combined with the G238S:E240K substitutions. In contrast, the A237G substitution acts additively with the G238S:E240K substitutions to increase aztreonam hydrolysis.
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PMID:Selection and characterization of amino acid substitutions at residues 237-240 of TEM-1 beta-lactamase with altered substrate specificity for aztreonam and ceftazidime. 879 21

Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor beta-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(L-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D.
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PMID:Insulin-induced tyrosine dephosphorylation of paxillin and focal adhesion kinase requires active phosphotyrosine phosphatase 1D. 880 54

Fluid shear stress modulates vascular function and structure by stimulating mechanosensitive endothelial cell signal events. Cell adhesion, mediated by integrin-matrix interactions, also regulates intracellular signaling by mechanosensitive events. To gain insight into the role of integrin-matrix interactions, we compared tyrosine phosphorylation and extracellular signal-regulated kinase (ERK1/2) activation in adhesion- and shear stress-stimulated human umbilical vein endothelial cells (HUVEC). Adhesion of HUVEC to fibronectin, but not to poly-L-lysine, rapidly activated ERK1/2. Fluid shear stress (12 dyn/cm2) enhanced ERK1/2 activation stimulated by adhesion, suggesting the presence of a separate pathway. Two differences in signal transduction were identified: focal adhesion kinase phosphorylation was increased rapidly by adhesion but not by shear stress; and ERK1/2 activation in response to adhesion was inhibited to a significantly greater extent when actin filaments were disrupted by cytochalasin D. Two similarities in activation of ERK1/2 were observed: protein kinase C (PKC) activity was necessary as shown by complete inhibition when PKC was downregulated; and an herbimycin-sensitive (genistein- and tyrphostin-insensitive) tyrosine kinase was required. c-Src was identified as a candidate tyrosine kinase as it was activated by both shear stress and adhesion. These findings suggest that adhesion and shear stress activate ERK1/2 via a shared pathway that involves an herbimycin-sensitive tyrosine kinase and PKC. In addition, shear stress activates ERK1/2 through another pathway that is partially independent of cytoskeletal integrity.
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PMID:Mitogen-activated protein kinase (ERK1/2) activation by shear stress and adhesion in endothelial cells. Essential role for a herbimycin-sensitive kinase. 895 27

An automatic and sensitive HPLC method for the determination of primary and secondary amino acids included cystine and homocystine in urine samples is described. After a simple ultrafiltration, urine samples were subjected to reduction of disulfides, carboxymethylation of free thiols and double precolumn derivatization with o-phthalaldehyde-3-mercaptopropionic acid and 9-fluroenylmethyl chloroformate. All reactions were fully automated by means of an injector programme and were accomplished in 10 min. Since urine samples contain a large number of amino compounds, a good resolution was required. By optimization of the conditions, separation of 40 amino acids in 92 min was achieved. The recovery of amino acids ranged from 83% for TRP to 105% for CIT. The within-run and between-run R.S.D.s of urinary amino acid concentrations were below 10% for most amino acids except for HYL, LYS and ORN. The method was applied to the diagnosis of genetic disorders of amino acid metabolism.
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PMID:Automated method for the measurement of amino acids in urine by high-performance liquid chromatography. 900 38


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