Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The biochemical and biophysical characteristics of Janus protein-tyrosine kinases (JAKs), which are essential early mediators of cytokine-initiated signal propagation, are virtually undefined. To facilitate the in vitro analysis of JAK-mediated catalysis, we substantially purified a soluble recombinant JAK2 and developed a novel means of quantifying JAK-catalyzed product formation. Glutathione-S-transferase fusion proteins containing active and inactive forms of rat Janus kinase 2 (GST:rJAK2 and GST:rJAK2(CA795)) were highly purified via affinity chromatography. A microtiterplate-based ELISA was used to measure tyrosine phosphorylation of a streptavidin-immobilized biotinylated STAT1-derived peptide. The ELISA data indicated that only about 1% of the enzyme was involved in exogenous substrate phosphorylation. Other immobilized peptides served as apparent substrates with varying efficacy. Traditional radioisotopic autokinase assays demonstrated that the activity of the purified fusion protein was inhibited by a variety of tyrphostin inhibitors. Non-radiolabeled adenine nucleotides, but not guanine nucleotides, inhibited the radioisotopic autokinase assay. These observations verify that the catalytic activity of JAK2 is highly regulated, and are consistent with the suggestion that JAK2 may require additional accessory proteins, such as a potential upstream regulatory kinase, for full catalytic activity.
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PMID:Characterization of the in vitro kinase activity of a partially purified soluble GST/JAK2 fusion protein. 1219 Jan 18

Recent study has shown that nuclear glutathione S-transferase (GST) pi accumulates in cancer cells resistant to doxorubicin hydrochloride (DOX) and may function to prevent nuclear DNA damage caused by DOX (Goto et al., FASEB J., 15, 2702 - 2714 (2001)). It is not clear if the amount of nuclear GSTpi increases in response to other anti-cancer drugs and if so, what is the physiological significance of the nuclear transfer of GSTpi in the acquisition of drug-resistance in cancer cells. In the present study, we employed three cancer cell lines, HCT8 human colonic cancer cells, A549 human lung adenocarcinoma cells, and T98G human glioblastoma cells. We estimated the nuclear transfer of GSTpi induced by the anti-cancer drugs cisplatin (CDDP), irinotecan hydrochloride (CPT-11), etoposide (VP-16) and 5-fluorouracil (5-FU). It was found that: (1) Nuclear GSTpi accumulated in these cancer cells in response to CDDP, DOX, CPT-11, VP-16 and 5-FU. (2) An inhibitor of the nuclear transport of GSTpi, edible mushroom lectin (Agaricus bisporus lectin, ABL), increased the sensitivity of the cancer cells to DOX and CDDP, and partially to CPT-11. Treatment with ABL had no apparent effect on the cytotoxicity of VP-16 and 5-FU. These results suggest that inhibitors of the nuclear transfer of GSTpi have practical value in producing an increase of sensitivity to DOX, CDDP and CPT-11.
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PMID:Significance of nuclear glutathione S-transferase pi in resistance to anti-cancer drugs. 1235 59

The anti-angiogenic agents angiostatin and endostatin have been shown to affect endothelial cell migration in a number of studies. We have examined the effect of these agents on intracellular signalling pathways known to regulate endothelial cell migration and proliferation/survival. Both agents inhibited fibroblast growth factor (FGF)-, and vascular endothelial growth factor (VEGF)-mediated migration of primary human microvascular endothelial cells and affected vascular formation in the embryoid body model. However, using phosphospecific antibodies we could not detect any effect of angiostatin or endostatin on phospholipase C-gamma (PLC-gamma), Akt/PKB, p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK and p21-activated kinase (PAK) activity. Furthermore, using a glutathione S-transferase (GST)-PAK pull-down assay, we could not detect any effect on Rac activity. We conclude that angiostatin and endostatin inhibit chemotaxis, without affecting intracellular signalling pathways known to regulate endothelial migration and proliferation/survival.
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PMID:Angiostatin and endostatin inhibit endothelial cell migration in response to FGF and VEGF without interfering with specific intracellular signal transduction pathways. 1258 31

Cleavage of the beta-aryl ether linkage is the most important process in lignin degradation. Here we characterize the three tandemly located glutathione S-transferase (GST) genes, ligF, ligE, and ligG, from low-molecular-weight lignin-degrading Sphingomonas paucimobilis SYK-6, and we describe the actual roles of these genes in the beta-aryl ether cleavage. Based on the identification of the reaction product by electrospray ionization-mass spectrometry, a model compound of beta-aryl ether, alpha-(2-methoxyphenoxy)-beta-hydroxypropiovanillone (MPHPV), was transformed by LigF or LigE to guaiacol and alpha-glutathionyl-beta-hydroxypropiovanillone (GS-HPV). This result suggested that LigF and LigE catalyze the nucleophilic attack of glutathione on the carbon atom at the beta position of MPHPV. High-pressure liquid chromatography-circular dichroism analysis indicated that LigF and LigE each attacked a different enantiomer of the racemic MPHPV preparation. The ligG gene product specifically catalyzed the elimination of glutathione from GS-HPV generated by the action of LigF. This reaction then produces an achiral compound, beta-hydroxypropiovanillone, which is further degraded by this strain. Disruption of the ligF, ligE, and ligG genes in SYK-6 showed that ligF is essential to the degradation of one of the MPHPV enantiomers, and the alternative activities which metabolize the substrates of LigE and LigG are present in this strain.
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PMID:Roles of the enantioselective glutathione S-transferases in cleavage of beta-aryl ether. 1261 39

CSK family contains two protein tyrosine kinases: Csk (C-terminal Src kinase) and Chk (Csk homologous kinase). They are responsible for phosphorylating Src family protein tyrosine kinases on a C-terminal Tyr (Tyr527) and negatively regulating their activities. However, Chk and Csk have different expression patterns, mechanisms of regulation, and different biological functions, and appear to play different roles in the development of breast cancer. To obtain pure human Chk for biochemical characterization, its coding region was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The enzyme was highly expressed but unusually prone to proteolytic degradation during purification. Expression of the enzyme as a dual fusion protein with glutathione S-transferase on N-terminus and streptag, a 10 amino acid peptide, on C-terminus allowed purification of the full-length fusion protein. The purified enzyme was able to phosphorylate and inactivate Src. Chk (no inhibition up to 18.5 microM) and Csk (IC(50)= 1 microM) were differentially inhibited by PP2, probably due to the size difference of one residue (Thr265 in Csk versus Met304 in Chk) in the ATP-binding domain. The expression, purification, and initial characterizations of Chk provided an important step toward full characterization of Chk and Csk, two important enzymes in cellular regulation.
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PMID:Expression, purification, and biochemical characterization of Chk, a soluble protein tyrosine kinase. 1276 3

CLCA (chloride channel, calcium-activated) proteins are novel pulmonary vascular addresses for blood-borne, lung-metastatic cancer cells. They facilitate vascular arrest of cancer cells via adhesion to beta4 integrin and promote early, intravascular, metastatic growth. Here we identify the interacting binding domains of endothelial CLCA proteins (e.g. hCLCA2, mCLCA5, mCLCA1, and bCLCA2) and beta4 integrin. Endothelial CLCAs share a common beta4-binding motif (beta4BM) in their 90- and 35-kDa subunits of the sequence F(S/N)R(I/L/V)(S/T)S, which is located in the second extracellular domain of the 90-kDa CLCA and near the N terminus of the 35-kDa CLCA, respectively. Using enzyme-linked immunosorbent, pull-down, and adhesion assays, we showed that glutathione S-transferase fusion proteins of beta4BMs from the 90- and 35-kDa CLCA subunits bind to the beta4 integrin in a metal ion-dependent manner. Fusion proteins from fibronectin and the integrins beta1 and beta3 served as negative controls. beta4BM fusion proteins competitively blocked the beta4/CLCA adhesion and prevented lung colonization of MDA-MB-231 breast cancer cells. A disrupted beta4BM in hCLCA1, which is not expressed in endothelia, failed to interact with beta4 integrin. The corresponding CLCA-binding domain of the beta4 integrin is localized to the specific determining loop (SDL). Again enzyme-linked immunosorbent, pull-down, and adhesion assays were used to confirm the interaction with CLCA proteins using a glutathione S-transferase fusion protein representing the C-terminal two-thirds of beta4 SDL (amino acids 184-203). A chimeric beta4 integrin in which the indicated SDL sequence had been replaced with the corresponding sequence from the beta1 integrin failed to bind hCLCA2. The dominance of the CLCA ligand in beta4 activation and outside-in signaling is discussed in reference to our previous report that beta4/CLCA ligation elicits selective signaling via focal adhesion kinase to promote metastatic growth.
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PMID:The interacting binding domains of the beta(4) integrin and calcium-activated chloride channels (CLCAs) in metastasis. 1451 19

SNARK, the fourth member of the AMPK catalytic subunit family, was originally identified in a rat kidney cDNA library, and in this study we isolated its human homologue. A BLAST search analysis using rat SNARK protein yielded a single high homology clone, DKFZp434J037, isolated from human testis, and since its hypothetical protein showed 84% homology to rat SNARK protein, we assumed DKFZp434J037 to be the human SNARK cDNA. The human SNARK cDNA is 3443bp long and encodes a 628 amino acid protein having an estimated molecular weight of 69kDa, and its chromosomal localization had been assigned to 1q32.1. The same as other members of AMPK catalytic subunit family, human SNARK showed AMP-dependent GST-SAMS phosphorylation activity and enhanced HepG2 cell survival during glucose starvation. Human SNARK-overexpressing HepG2 cells (H/SNK) showed acute cell-cell detachment when exposed to glucose-free medium and the cell-cell detachment correlated well with the detection of G-actin. Deletion mutant analysis strongly suggested that the putative catalytic domain of SNARK is necessary for the cell-cell detachment, and Western blotting analysis showed that phosphorylation of FAK and PKC, which were dramatically increased by glucose starvation in HepG2 cells, was markedly suppressed by SNARK.
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PMID:Induction of cell-cell detachment during glucose starvation through F-actin conversion by SNARK, the fourth member of the AMP-activated protein kinase catalytic subunit family. 1457 7

Previous studies have shown that the adaptor protein Shb is involved in receptor tyrosine kinase signaling. In this study, we demonstrate that Shb is phosphorylated in an Src-dependent manner upon vascular endothelial growth factor (VEGF) stimulation using porcine aortic endothelial cells expressing the human VEGF receptor 2 (VEGFR-2) (KDR). In co-immunoprecipitation experiments, we could detect an interaction between Shb and the VEGFR-2 in human telomerase-immortalized microvascular endothelial cells. Furthermore, in a glutathione S-transferase pull-down assay, the Src homology 2 domain of Shb was shown to interact with phosphorylated tyrosine 1175 in the C-terminal tail of VEGFR-2. VEGF-induced Shb phosphorylation was lost in porcine aortic endothelial cells expressing a chimeric murine VEGFR-2 (Flk-1) with a mutation at the corresponding position. Shb expression was specifically decreased by 80%, in a transient manner, by using the short interfering RNA technique. Reduced Shb expression led to a loss of stimulation of phosphatidylinositol 3-kinase, phosphorylation of focal adhesion kinase at tyrosine 576, the generation of focal adhesions, and stress fiber formation in response to VEGF. Furthermore, we show that VEGF-induced migration is inhibited in Shb short interfering RNA-treated cells. Our data demonstrate that Shb is important for VEGF signaling in endothelial cells. This is achieved by Shb binding to tyrosine 1175 in the VEGFR-2, which regulates VEGF-induced formation of focal adhesions and cell migration, of which the latter occurs in a phosphatidylinositol 3-kinase-dependent manner.
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PMID:The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration. 1502 17

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)), controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the Vitamin D receptor (VDR) interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the Vitamin D receptor. However, the relationship between these two classes of coactivators is not clear. Using GST-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP205 and SRC-3 potentiated Vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are differentially involved in Vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.
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PMID:Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in VDR transactivation during keratinocyte differentiation. 1522 84

Cross-talk between ERalpha and STAT5a was demonstrated to mediate through a direct physical association between the two proteins. By GST pull-down assays and functional assays with various constructs of ERalpha and STAT5a, it was shown that the C-termini of these two proteins were mainly responsible for this interaction. Furthermore, the interaction between ERalpha and STAT5a was demonstrated to give rise to functional changes in their signaling events. In cell transfection studies, it was shown that ERalpha activation could attenuate PRLR signaling through STAT5a. This ERalpha-mediated attenuation of PRLR signaling was substantiated by observed decreases in the phosphorylation of JAK2 and STAT5a, reduced translocation of STAT5a into the nucleus, and reduced binding of STAT5a onto a GAS-containing nucleotide. Apart from transfected cells, the interaction between ERalpha and STAT5a could also be observed in established breast cancer cell lines of MCF-7 and T-47D in co-immunoprecipitation studies. However, the functional consequence of the interaction in these cancer cells was very different from the transfected HEK293 cells. ER activation could lead to potentiation of PRLR signaling in MCF-7 cells but not in T-47D cells. Conversely, in both MCF-7 and T-47D cells, PRLR activation could lead to attenuation of ER signaling. These data serve to elucidate the mechanisms underlying the ERalpha-STAT5a cross-talk and in demonstrating that the functional consequence of this cross-talk depends on the precise milieus of the intracellular environment.
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PMID:ERalpha and STAT5a cross-talk: interaction through C-terminal portions of the proteins decreases STAT5a phosphorylation, nuclear translocation and DNA-binding. 1530 55


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