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)

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

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

T cell receptor zeta (TcRzeta)/CD3 ligation initiates a signaling cascade that involves src kinases p56(lck) and zeta-associated protein 70, leading to the phosphorylation of substrates such as TcRzeta, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59(fyn), the TcRzeta/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59(fyn), FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRzeta/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.
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PMID:Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production. 920 19

Uptake of L-[14C]glutamate (L-[14C]GLU) into nonsynaptic mitochondria isolated from rat cerebral hemispheres was measured in the presence of potential modulators of amino acid transport. The L-GLU carrier agonist 0.2 mM L-aspartate (L-ASP) virtually abolished L-GLU uptake (ASP/GLU concentration ratio, 1:1). L-Arginine (L-ARG) inhibited L-GLU uptake in a dose dependent manner over the concentration range 0.1-5 mM to maximum inhibition of 85%. Putrescine or ammonia had no effect, whereas 5 mM creatine and the NO generator, 5 mM sodium nitroprusside, increased the uptake by 73% and 57%, respectively. D-ARG was three times less effective in inhibiting L-GLU uptake than L-ARG at 5 mM concentration. The L-amino acids ornithine, lysine, histidine, tyrosine, phenylalanine, proline, leucine, isoleucine, tryptophan, glycine, methionine, valine, serine, taurine, alanine or cysteine did not affect the uptake when added in concentrations of 2-5 mM. A 14% inhibition of L-GLU uptake was noted in the presence of L-glutamine (L-GLN) (2 mM) or a dicarboxylate carrier ligand, alpha-ketoglutarate (alpha-KG) (5 mM), and a 30% inhibition with a dicarboxylate carrier inhibitor phenylsuccinate (PhSc) (5 mM). The results suggest that L-ARG functions as a specific endogenous modulator of cerebral mitochondrial L-GLU transport.
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PMID:Glutamate uptake is inhibited by L-arginine in mitochondria isolated from rat cerebrum. 924 41

Four Holstein steers (159 kg) surgically fitted with abomasal-infusion cannulas were used in a 4 x 4 Latin square study to test amino acid (AA) and casein (CAS) infusions on nitrogen balance and hormonal status of steers consuming vegetative wheat (Triticum aestivum L.) silage (12.3% CP). Treatments were 5-d infusions of 1) water (CONT), 2) arginine (ARG; 13.69 g/d), 3) limiting amino acids (LAA, 13.69 g/d arginine + 10.92 g/d histidine + 28.97 g/d lysine + 10.88 g/d methionine + 16.96 g/d threonine, and 4) Na-CAS (300 g/d). Whole blood was collected for plasma AA, growth hormone (GH), insulin, and IGF-I concentrations. Data were analyzed by ANOVA, and the following orthogonal contrasts were used to separate treatment means: CONT vs ARG; ARG vs LAA; and LAA vs CAS. Urinary N increased (P < .02) for CAS vs LAA. Arginine increased N retention, as did CAS, compared to LAA. Total plasma essential AA were decreased by arginine. Mean plasma insulin concentrations were increased by CAS (P < .034). Arginine increased mean plasma GH levels, but not IGF-I. The CAS treatment increased (P < .015) IGF-I levels, but not GH. These data suggest that performance of steers fed wheat silage was limited by duodenal AA flow and that arginine was the first-limiting AA. Casein infusion increased plasma insulin and IGF-I, which would explain the improved growth noted in calves and lambs fed forages supplemented with ruminally undegraded protein.
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PMID:Nitrogen metabolism and hormonal responses of steers fed wheat silage and infused with amino acids or casein. 937 20

We have investigated the influence of the nitric oxide synthase (NOS) substrate, NG-hydroxy-L-arginine (H-ARG) on dopamine (DA) and glutamate (GLU) efflux in vivo using concentric microdialysis probes implanted in the anterior-medial striatum of chloral hydrate-anesthetized rats. Intrastriatal infusion of H-ARG (100 microM, 200 microM, or 1 mM for 120 min) increased DA efflux in a dose-dependent fashion. The facilitatory effect of H-ARG (1 mM) on DA efflux was abolished following pretreatment (80 min) with the constitutive NOS inhibitor 7-nitroindazole (7-NI, 10 microM) but unaffected by L-NG(1-iminoethyl) lysine (100 microM) infusion. As both H-ARG (1 mM) and the NO-generator (+/-)-S-nitroso-N-acetylpenicillamine (1 mM) were observed to increase GLU efflux concurrently with the effect on DA efflux, we evaluated the potential intermediary role of GLU in NO-facilitated DA efflux using ionotropic GLU receptor antagonists. Local infusion of dizocilpine maleate (10 microM) or (+/-)-2-amino-3-[3-(carboxymethoxy)-5-methyl-isoxazol-4-yl] propionic acid (100 microM), attenuated the H-ARG (1 mM)-induced elevation of extracellular DA levels. Conversely, similar treatment with the kainate receptor antagonist d-gamma-glutamyl-aminomethanesulfonic acid did not alter H-ARG-induced DA efflux. To evaluate the regulatory influence of striatal NO on NMDA receptor activation, NMDA (100 microM) was co-perfused with either H-ARG (2 mM) or 7-NI (10 microM). While co-perfusion with 7-NI potentiated NMDA-induced DA efflux, similar treatment with H-ARG (2 mM) abolished the effect. These results demonstrate that endogenous NO production, stimulated via H-ARG-dependent activation of type 1 NOS, enhances striatal DA efflux via an increase in glutamatergic tone on ionotropic GLU-receptors. At higher levels of NOS activation (following H-ARG (2 mM) or NMDA infusion), NO may block glutamatergic neurotransmission via inhibition of NMDA receptor function.
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PMID:Endogenous nitric oxide facilitates striatal dopamine and glutamate efflux in vivo: role of ionotropic glutamate receptor-dependent mechanisms. 951 28

The Saccharomyces cerevisiae a1 homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium. Nuclear magnetic resonance (NMR) spectra of previously prepared a1 homeodomain samples contained a subset of doubled and broadened resonances. Mass spectroscopic and NMR analysis demonstrates that the heterogeneity is largely due to a lysine misincorporation at the arginine (Arg) 115 site. Arg 115 is coded by the 5'-AGA-3' sequence, which is quite rare in E. coli genes. Lower level mistranslation at three other rare arginine codons also occurs. The percentage of lysine for arginine misincorporation in a1 homeodomain production is dependent on media composition. The dnaY gene, which encodes the rare 5'-AGA-3' tRNA(ARG), was co-expressed in E. coli with the a1-encoding plasmid to produce a homogeneous recombinant a1 homeodomain. Co-expression of the dnaY gene completely blocks mistranslation of arginine to lysine during a1 overexpression in minimal media, and homogeneous protein is produced.
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PMID:High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae a1 homeodomain expressed in Escherichia coli. 952 Nov 27

Transmission of zucchini yellow mosaic virus (ZYMV) by aphids was examined by introducing mutations within the highly conserved proline-threonine-lysine (PTK) motif of the helper component proteinase (HC-Pro) using a cDNA full-length clone. Replacement of proline by alanine (ATK) in the PTK motif abolished transmission almost completely both from plants and from membranes. Substitution of the basic lysine by glutamic acid (PTE) did not reduce the rate of transmission compared with the wild-type. Replacement of threonine by valine (PVK) or serine (PSK) resulted in a rate of transmission that was lower than that of the wild-type. The rate was lower for PSK than for PVK. Western blot comparison did not permit attribution of HC-Pro functionality in transmission to its level in the host. The HC-Pro of strains that effected transmission (with the wild-type PTK motif, and with the mutated PTE and PVK motifs) could also bind in vitro to virions of ZYMV. HC-Pro with a PSK motif, which was less effective in assisting transmission, could bind only weakly to virions, while HC-Pro of the almost non-transmissible strains (with PAK and ATK motifs) did not bind at all. Interestingly, positive binding was recorded for transmission-defective ZYMV-Ct, which has a PTK motif but has glutamic acid instead of lysine in the lysine-leucine-serine-cysteine (KLSC) motif. These findings support the 'bridge hypothesis', and confirm the binding of the HC-Pro to the virion. The possible role of the PTK and KLSC motifs in binding to the virus and to the mouthparts of the aphid is discussed.
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PMID:Mutations in the HC-Pro gene of zucchini yellow mosaic potyvirus: effects on aphid transmission and binding to purified virions. 956 86

The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.
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PMID:Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration. 977 38


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