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)

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

Mutations of the Janus family kinase JAK3 have been found to be responsible for autosomal recessive severe combined immunodeficiency (SCID) in humans. We report here the analysis of four new unrelated patients affected by JAK3-deficient SCID. The genetic defects were heterogeneous and included a large intragenic deletion as well as different point mutations, leading to missense substitutions, early stop codons, or splicing defects. We performed a series of studies of the biochemical events induced by cytokines on lymphoblastoid B-cell lines obtained from these patients. Abnormalities in tyrosine phosphorylation of JAK3 in response to interleukin-2 (IL-2) and IL-4 were present in all patients. Accordingly, IL-2-mediated phosphorylation of STAT5 was also absent or barely detectable. On the contrary, in all cases, we could show reduced but clear phosphorylation of STAT6 upon IL-4 stimulation. In one patient carrying a single amino acid change (Glu481Gly) in the JH3 domain of JAK3, we observed partially conserved IL-2 responses resulting in reduced but detectable levels of JAK3 and STAT5 phosphorylation. Interestingly, the patient bearing this mutation developed a substantial number of circulating CD4(+)/CD45RO+ activated T lymphocytes that were functionally impaired. In two cases, patients' cells expressed JAK3 proteins with mutations in the JH2 pseudo-kinase domain. A single cysteine to arginine substitution (Cys759Arg) in this region resulted in high basal levels of constitutive JAK3 tyrosine phosphorylation unresponsive to either downregulation by serum starvation or cytokine-mediated upregulation. The characterization of the genetic defects and biochemical abnormalities in these JAK3-deficient patients will help define the role of JAK3 in the ontogeny of a competent immune system and may lead to a better understanding of the JAK3 functional domains.
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PMID:Structural and functional basis for JAK3-deficient severe combined immunodeficiency. 935 68

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 EWS/FLI1 fusion gene found in Ewing's sarcoma and primitive neuroectodermal tumor, is able to transform certain cell lines by acting as an aberrant transcription factor. The ability of EWS/FLI1 to modulate gene expression in cells transformed and resistant to transformation by EWS/FLI1, was assessed by Representational Difference Analysis (RDA). We found that the cyclin selective ubiquitin conjugase murine E2-C, was up regulated in NIH3T3 cells transformed by EWS/FLI1 but not in a nontransformed NIH3T3 clone expressing EWS/FLI1. We also found that mE2-C is upregulated in NIH3T3 cells transformed by other genes including activated cdc42, v-ABL and c-myc. We demonstrated that expression of mE2-C in both the EWS/FLI1 transformed and parent NIH3T3 lines varies with the cell cycle. Finally, dominant-negative mE2-C, created by changing a catalytic cysteine to serine, inhibits the in vitro ubiquitination and degradation of cyclin B in human HeLa cell extracts. These data suggest that part of the biologic effect of EWS/FLI1 could be to transcriptionally modulate genes involved in cell cycle regulation.
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PMID:EWS/FLI1 up regulates mE2-C, a cyclin-selective ubiquitin conjugating enzyme involved in cyclin B destruction. 979 75

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinson's disease, pulmonary fibrosis, and Alzheimer's disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c-fos and c-myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2 stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-L-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation.
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PMID:Activation of the JAK-STAT pathway by reactive oxygen species. 984 26

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds with guaiacyl moieties, which are converted into protocatechuate by the actions of lignin degradation enzymes in this strain. Protocatechuate is a key metabolite in the SYK-6 degradation of lignin compounds with guaiacyl moieties, and it is thought that it degrades to pyruvate and oxaloacetate via the protocatechuate 4,5-cleavage pathway. In a 10.5-kb EcoRI fragment carrying the protocatechuate 4,5-dioxygenase gene (ligAB) (Y. Noda, S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. J. Bacteriol. 172:2704-2709, 1990), we found the ligI gene encoding 2-pyrone-4, 6-dicarboxylic acid (PDC) hydrolase. PDC hydrolase is a member of this pathway and catalyzes the interconversion between PDC and 4-carboxy-2-hydroxymuconic acid (CHM). The ligI gene is thought to be transcribed divergently from ligAB and consists of an 879-bp open reading frame encoding a polypeptide with a molecular mass of 32,737 Da. The ligI gene product (LigI), expressed in Escherichia coli, was purified to near-homogeneity and was estimated to be a monomer (31.6 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9. The optimum pH for hydrolysis of PDC is 8.5, the optimum pH for synthesis of PDC is 6.0 to 7.5, and the Km values for PDC and CHM are 74 and 49 microM, respectively. LigI activity was inhibited by the addition of thiol reagents, suggesting that the cysteine residue is a catalytic site. LigI is more resistant to metal ion inhibition than the PDC hydrolases of Pseudomonas ochraceae (K. Maruyama, J. Biochem. 93:557-565, 1983) and Comamonas testosteroni (P. J. Kersten, S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lipscomb, J. Bacteriol. 152:1154-1162, 1982). The insertional inactivation of the ligI gene in S. paucimobilis SYK-6 led to the complete loss of PDC hydrolase activity and to a growth defect on vanillic acid; it did not affect growth on syringic acid. These results indicate that the ligI gene is essential for the growth of SYK-6 on vanillic acid but is not responsible for the growth of SYK-6 on syringic acid.
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PMID:Genetic and biochemical characterization of a 2-pyrone-4, 6-dicarboxylic acid hydrolase involved in the protocatechuate 4, 5-cleavage pathway of Sphingomonas paucimobilis SYK-6. 986 12

Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR.
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PMID:rlk/TXK encodes two forms of a novel cysteine string tyrosine kinase activated by Src family kinases. 989 Oct 83

PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways.
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PMID:The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells. 1002 29

Caspases, a family of cysteine proteases, are the key effector proteins of apoptosis. These proteases cleave cellular proteins and are responsible for the destruction of the cell body during apoptosis. They are also involved in the activation of other proteins, such as cytokines. In this study, we demonstrate a novel function for these proteases. Z-Asp-CH2-DCB (Z-Asp), a general caspase inhibitor, blocked cell spreading on collagen-coated plates in a dose-dependent manner but did not affect cell viability. Caspase 3-like activity but not caspase 1-like activity was detected in adherent cells on both collagen-coated and poly-L-lysine-coated plates but not in suspended cells. The caspase 3-like activity was significantly inhibited by Z-Asp. However, only Z-Asp, not specific caspase inhibitors (Z-DEVD for caspase 3, Z-YVAD for caspase 1), was effective in the suppression of cell spreading. The inhibitory effect of Z-Asp was blocked by a phosphokinase C activator, PMA, and a Rho activator, lysophosphatidic acid (LPA), while neither a Rac activator, bradykinin, nor a Cdc42 activator, sphingosine-1 -phosphate, was effective. Immunoprecipitation demonstrated that Z-Asp downregulated the expression of focal adhesion kinase (FAK) protein, downstream of Rho signaling, in adherent cells. Our results suggest that not caspase 1 or 3 but another yet unknown caspase(s) plays an important role in the maintenance of cytoskeleton integrity via FAK protein expression, implying a new function for caspases.
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PMID:Possible involvement of caspase-like family in maintenance of cytoskeleton integrity. 1008 31

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, alphaIIbbeta3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of alphaIIbbeta3. One clone (AM-1) expressed constitutively active alphaIIbbeta3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antialphaIIbbeta3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of beta3(T562N) was responsible for receptor activation. In fact, T562N also activated alphaVbeta3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated alphaIIbbeta3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of beta3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to alphaIIbbeta3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of beta3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.
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PMID:A mutation in the extracellular cysteine-rich repeat region of the beta3 subunit activates integrins alphaIIbbeta3 and alphaVbeta3. 1019 35


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