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)

We previously demonstrated that in white fat cell precursors alpha2-adrenoceptor stimulation lead to the phosphorylation of p44 and p42 mitogen-activated protein kinases and an increase in cell number. Regulation of cell adhesion and cell cytoskeleton plays a crucial role in the control of cell growth by various growth factors. Here, we report that in mouse 3T3F442A preadipocytes expressing 2500 fmol/mg protein of the human alpha2C10-adrenoceptor (alpha2AF2 cells), alpha2-adrenergic stimulation rapidly restored the spreading of cells previously retracted by serum withdrawal. This effect was pertussis toxin sensitive and was blocked by pretreatment of the cells with dihydrocytochalasin B (a blocker of actin polymerization), genistein (a tyrosine kinase inhibitor), or agents that increase cell cAMP content. Spreading was accompanied by cell membrane ruffling, formation of lamelipodia and filipodia, appearance of focal adhesion plaques, and induction of actin stress fibers. alpha2-Adrenergic stimulation also lead to a rapid Gi- and actin-dependent tyrosine phosphorylation of the pp125 focal adhesion kinase (FAK) as well as of the p42 and p44 mitogen-activated protein kinases. alpha2-Adrenergic-dependent spreading and FAK and mitogen-activated protein kinase phosphorylation were also observed in 3T3F442A preadipocytes permanently expressing 20 fmol/mg protein of the human alpha2C10-adrenoceptor (alpha2AF3 cells) as well as in BFC-1beta preadipocytes, which constitutively express 25 fmol/mg protein of mouse alpha2A-adrenoceptors. In BFC-1beta preadipocytes, alpha2-adrenergic-dependent spreading and pp125FAK phosphorylation were counteracted by beta-adrenergic stimulation. Our results suggest that alpha2-adrenergic control of actin polymerization and focal adhesion assembly could play a crucial role in the regulation of preadipocyte growth by the sympathetic nervous system.
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PMID:alpha2-Adrenoceptor stimulation promotes actin polymerization and focal adhesion in 3T3F442A and BFC-1beta preadipocytes. 894 Mar 38

Protein tyrosine phosphorylation, modulated by the rate of both protein tyrosine kinase and protein tyrosine phosphatase activities, is critical for cellular signal transduction cascades. We report that endothelin-1 stimulation of rabbit platelets resulted in a dose- and time-dependent tyrosine phosphorylation of four groups of proteins in the molecular mass ranges of 50, 60, 70-100 and 100-200 kDa and that one of these corresponds to focal adhesion kinase. This effect is also related to the approximately 60% decrease in protein tyrosine phosphatase activity. Moreover, this inhibited activity was less sensitive to orthovanadate. In the presence of forskolin that increases the cAMP level a dose-dependent inhibition of the endothelin-stimulated tyrosine phosphorylation of different protein substrates and a correlation with an increase in the protein tyrosine phosphatase activity (11.6-fold compared to control) have been found. Further studies by immunoblotting of immunoprecipitated soluble fraction with anti-protein tyrosine phosphatase-1C from endothelin-stimulated platelets have demonstrated that the tyrosine phosphorylation of platelet protein tyrosine phosphatase-1C is correlated with the decrease in its phosphatase activity. As a consequence, modulation and regulation by endothelin-1 in rabbit platelets can be proposed through a cAMP-dependent pathway and a tyrosine phosphorylation process that may affect some relevant proteins such as focal adhesion kinase.
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PMID:Protein tyrosine phosphatase activity modulation by endothelin-1 in rabbit platelets. 900 14

In rat liver epithelial cells (GN4), angiotensin II (Ang II) and thapsigargin stimulate a novel calcium-dependent tyrosine kinase (CADTK) also known as PYK2, CAKbeta, or RAFTK. Activation of CADTK by a thapsigargin-dependent increase in intracellular calcium failed to stimulate the extracellular signal-regulated protein kinase pathway but was well correlated with a 30-50-fold activation of c-Jun N-terminal kinase (JNK). In contrast, Ang II, which increased both protein kinase C (PKC) activity and intracellular calcium, stimulated extracellular signal-regulated protein kinase but produced a smaller, less sustained, JNK activation than thapsigargin. 12-O-Tetradecanoylphorbol 13-acetate (TPA), which slowly activated CADTK, did not stimulate JNK. These findings suggest either that CADTK is not involved in JNK activation or PKC activation inhibits the CADTK to JNK pathway. A 1-min TPA pretreatment of GN4 cells inhibited thapsigargin-dependent JNK activation by 80-90%. In contrast, TPA did not inhibit the >50-fold JNK activation effected by anisomycin or UV. The consequence of PKC-dependent JNK inhibition was reflected in c-Jun and c-Fos mRNA induction following treatment with thapsigargin and Ang II. Thapsigargin, which only minimally induced c-Fos, produced a much greater and more prolonged c-Jun response than Ang II. Elevation of another intracellular second messenger, cAMP, for 5-15 min also inhibited calcium-dependent JNK activation by approximately 80-90% but likewise had no effect on the stress-dependent JNK pathway. In summary, two pathways stimulate JNK in cells expressing CADTK, a calcium-dependent pathway modifiable by PKC and cAMP-dependent protein kinase and a stress-activated pathway independent of CADTK, PKC, and cAMP-dependent protein kinase; the inhibition by PKC can ultimately alter gene expression initiated by a calcium signal.
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PMID:Protein kinase C and protein kinase A inhibit calcium-dependent but not stress-dependent c-Jun N-terminal kinase activation in rat liver epithelial cells. 916 74

The aim of this study was to investigate whether the stimulatory effect of growth hormone (GH) on the in vitro maturation and cumulus expansion of bovine oocytes is exerted through the cAMP or the tyrosine kinase pathway. Therefore bovine cumulus-oocyte complexes (COCs) were cultured in Medium 199 without fetal calf serum and gonadotropins, but supplemented with 100 ng/ml bovine GH (bGH; NIH-GH-B18) with or without 10 microM methyl 2,5-dihydroxycinnamate (erbstatin analogue), a specific tyrosine kinase inhibitor; 100 microM 2',3'-dideoxyadenosine (DDA), a specific adenylate cyclase inhibitor; or 10 microM H-89, a specific inhibitor of cAMP-dependent protein kinase A. Epidermal growth factor (EGF; 20 ng/ml) was added as a positive control for tyrosine kinase activation, and FSH (0.05 IU/ml) was added as a positive control for cAMP mediation during in vitro maturation in the absence or presence of the inhibitors. Culture was performed at 39 degrees C in a humidified atmosphere with 5% CO2 in air. To assess the effect on nuclear maturation, the proportion of oocytes in metaphase II stage after 16 h of culture was determined using 4,6-diamino-2-phenylindole staining. To determine the effect on cumulus expansion, the diameter of COCs at the onset and after 24 h of culture was measured. The stimulatory effects of GH on oocyte maturation and cumulus expansion were blocked by DDA and H-89 (p < 0.01). Similarly, FSH-induced cumulus expansion was abolished by DDA and H-89 (p < 0.05), while DDA did not block either EGF-induced oocyte maturation or cumulus expansion. Erbstatin analogue significantly blocked the stimulation of oocyte maturation and cumulus expansion by EGF (p < 0.02) but did not inhibit GH action on the COCs. It is concluded that the stimulatory effect of GH on oocyte maturation and cumulus expansion is mediated by the cAMP signal transduction pathway and not by JAK2 phosphorylation.
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PMID:Stimulatory effect of growth hormone on in vitro maturation of bovine oocytes is exerted through the cyclic adenosine 3',5'-monophosphate signaling pathway. 940 58

Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100ng/mal IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 microg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAK1 and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.
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PMID:Effect of recombinant interleukin-6 and thrombopoietin on isolated guinea pig bone marrow megakaryocyte protein phosphorylation and proplatelet formation. 941 Apr 69

Lipoprotein lipase (LPL) is important in the process of triglyceride storage in adipose tissue. Depression of LPL activity in adipose tissue is associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced wasting syndrome and may have a role in the associated serum hyperlipidemia produced by TCDD. The 3T3-L1 cell line was used as an in vitro model, independent of hormonal, nutritional, or other interfering factors associated with in vivo studies, in order to systematically examine the mechanism of action of TCDD. TCDD produced a statistically significant (P < 0.05) time- and dose-dependent decrease in LPL activity. Results of experiments with Ah-receptor blockers and structure activity studies with different polychlorinated biphenyl (PCB) and dioxin congeners were consistent with reduction of LPL activity being mediated by the Ah receptor. Culturing of 3T3-L1 cells without glucose or with cytochalasin B, a blocker of facilitative glucose transporters (GLUT), was effective in reducing LPL activity (P < 0.05). TCDD did not further reduce LPL activity in cytochalasin B pretreated 3T3-L1 cells or in 3T3-L1 cells cultured in glucose-free media. Dexamethasone pretreatment, which is known to increase GLUT expression in 3T3-L1 cells, prevented the reduction of LPL activity by TCDD. Protein tyrosine kinase activities, assayed using gamma-32P-ATP and RR-SRC, a src specific peptide substrate, were significantly increased (P < 0.05) over control levels by both TCDD and glucose deprivation. Furthermore, results of experiments treating 3T3-L1 cells with either insulin, EGF, 8-Br-cAMP, TPA, or genistein, alone or in combination with TCDD, were generally consistent with the hypothesis that lowered intracellular glucose and altered cellular kinase activities may be involved in reduction of LPL activities by TCDD. Further work is needed to confirm and better understand the role protein phosphorylation plays in TCDD-mediated alteration of glucose disposition and LPL activity. In summary, TCDD reduced LPL activity in 3T3-L1 cells as seen in vivo. Manipulation of glucose transport through a number of experimental approaches produced changes in 3T3-L1 LPL activity consistent with results of previous investigators showing glucose to be a positive regulator of LPL activity and consistent with our hypothesis that TCDD-mediated reduction of glucose transport is an important factor in the down regulation of LPL activity by TCDD.
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PMID:2,3,7,8-Tetrachlorodibenzo-p-dioxin mechanism of action to reduce lipoprotein lipase activity in the 3T3-L1 preadipocyte cell line. 941 85

Recent reports show that alpha-MSH (melanocyte-stimulating hormone) is mitogenic and melanogenic for normal human melanocytes, and that this effect is mediated through binding to the melanocortin receptor (MC1R) and activation of cAMP formation. alpha-MSH has also been shown to induce changes in cell shape in melanocytes and melanoma cells, particularly increased dendricity, suggesting a potential role for alpha-MSH in melanocyte-matrix interactions and pigment transfer through reorganization of the melanocyte actin filament cytoskeleton. In this report we show that the potent alpha-MSH analog (Nle4, D-Phe7)-alpha-MSH (NDP-MSH) induces reorganization of the actin stress fiber cytoskeleton in treated human melanocytes and that this reorganization is associated with increased adhesion to fibronectin (FN). Because most melanocyte growth factors act synergistically on melanocyte mitogenesis, we also sought to determine the effect of the melanocyte mitogen endothelin-1 (ET-1) on the melanocyte actin cytoskeleton, melanocyte adhesion, and melanocyte migration. We show that ET-1, which increases melanocyte migration on FN, has opposite effects on melanocyte adhesion to FN compared with NDP-MSH and that endothelin-1-induced actin reorganization is distinct from that observed following NDP-MSH treatment. Finally, we show that focal adhesion kinase (pp125FAK), a nonreceptor tyrosine kinase associated with focal contact formation and cell migration, is phosphorylated on tyrosine residues after treatment of melanocytes with ET-1, but not NDP-MSH. These data indicate that while alpha-MSH and ET-1 act synergistically to modulate melanocyte proliferation, they have opposite effects on melanocyte-matrix interactions.
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PMID:Alpha-melanocyte-stimulating hormone and endothelin-1 have opposing effects on melanocyte adhesion, migration, and pp125FAK phosphorylation. 941 62

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed cells were isolated and passaged. They multiplied at 33 C and formed multilayers. At the nonpermissive temperature (40 C), cells reverted to the normal differentiated phenotype similar to the primary luteal cells in culture. To determine whether GG-CL cells express the genes found in normal luteal cells, messenger RNA (mRNA) expression was examined by either Northern analysis or RT-PCR with primers specific to each mRNA. GG-CL cells were found to express receptors for interleukin-6 and glucocorticoid, as well as the newly discovered estrogen receptor-beta (ER-beta) and the orphan nuclear receptor nur 77. No receptors for ER-alpha, progesterone, LH, or PRL could be detected. This cell line also expressed 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), but not cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase, or aromatase cytochrome P450 (P450arom). Although the cells did not express the PRL receptor, they did express Janus kinase (JAK2) and signal transducers and activators of transcription (Stat5b), and, when transfected with the PRL receptor, they responded to PRL with a marked inhibition in 20alpha-HSD mRNA expression. In addition, estradiol enhanced ER-beta expression in a dose-dependent manner whereas cAMP stimulation caused a marked and rapid increase in the expression of the orphan receptor nur 77. In summary, a temperature-sensitive cell line was successfully established from the large luteal cells of rat corpora lutea. These cells express key genes encoding enzymes and receptors inherent to this defined luteal cell population and respond to stimulation by PRL, estradiol, and cAMP.
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PMID:Establishment and characterization of a simian virus 40-transformed temperature-sensitive rat luteal cell line. 952 80

Protein Phosphatase-1 (PP-1) appears to be the key component of the insulin signalling pathway which is responsible for bridging the initial insulin-simulated phosphorylation cascade with the ultimate dephosphorylation of insulin sensitive substrates. Dephosphorylations catalyzed by PP-1 activate glycogen synthase (GS) and simultaneously inactivate phosphorylase a and phosphorylase kinase promoting glycogen synthesis. Our in vivo studies using L6 rat skeletal muscle cells and freshly isolated adipocytes indicate that insulin stimulates PP-1 by increasing the phosphorylation status of its regulatory subunit (PP-1G). PP-1 activation is accompanied by an inactivation of Protein Phosphatase-2A (PP-2A) activity. To gain insight into the upstream kinases that mediate insulin-stimulated PP-1G phosphorylation, we employed inhibitors of the ras/MAPK, PI3-kinase, and PKC signalling pathways. These inhibitor studies suggest that PP-1G phosphorylation is mediated via a complex, cell type specific mechanism involving PI3-kinase/PKC/PKB and/or the ras/MAP kinase/Rsk kinase cascade. cAMP agonists such as SpcAMP (via PKA) and TNF-alpha (recently identified as endogenous inhibitor of insulin action via ceramide) block insulin-stimulated PP-1G phosphorylation with a parallel decrease of PP-1 activity, presumably due to the dissociation of the PP-1 catalytic subunit from the regulatory G-subunit. It appears that any agent or condition which interferes with the insulin-induced phosphorylation and activation of PP-1, will decrease the magnitude of insulin's effect on downstream metabolic processes. Therefore, regulation of the PP-1G subunit by site-specific phosphorylation plays an important role in insulin signal transduction in target cells. Mechanistic and functional studies with cell lines expressing PP-1G subunit site-specific mutations will help clarify the exact role and regulation of PP-1G site-specific phosphorylations on PP-1 catalytic function.
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PMID:Protein phosphatase-1 and insulin action. 960 13

This review summarizes the evolution of ideas concerning insulin signal transduction, the current information on protein ser/thr kinase cascades as signalling intermediates, and their status as participants in insulin regulation of energy metabolism. Best characterized is the Ras-MAPK pathway, whose input is crucial to cell fate decisions, but relatively dispensable in metabolic regulation. By contrast the effectors downstream of PI-3 kinase, although less well elucidated, include elements indispensable for the insulin regulation of glucose transport, glycogen and cAMP metabolism. Considerable information has accrued on PKB/cAkt, a protein kinase that interacts directly with Ptd Ins 3'OH phosphorylated lipids, as well as some of the elements further downstream, such as glycogen synthase kinase-3 and the p70 S6 kinase. Finally, some information implicates other erk pathways (e.g. such as the SAPK/JNK pathway) and Nck/cdc42-regulated PAKs (homologs of the yeast Ste 20) as participants in the cellular response to insulin. Thus insulin recruits a broad array of protein (ser/thr) kinases in its target cells to effectuate its characteristic anabolic and anticatabolic programs.
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PMID:Insulin signal transduction through protein kinase cascades. 960 12


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