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)

Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized after a 24-h treatment with 1alpha,25 dihydroxyvitamin D(3). The quantitative RT-PCR determined the mRNA levels of signaling molecules upstream and downstream Ras. The small GTPase is activated by guanine nucleotide exchange protein (GEF) and deactivated by GTPase-activating protein (GAP). When external stimuli are transduced into intracellular signals, various pathways are recruited: focal adhesion kinase (FAK) is associated with integrin-beta, and directs tyrosine phosphorylation of downstream substrates, including phospholipase C-gamma (PLC-gamma) and son of sevenless (SOS, a Ras GEF). The mRNA levels of FAK and PLC-gamma1 and -gamma2 in the flight cultures were increased 150% and 250% of the ground controls. The SOS mRNA levels in the flight cultures were increased 520% and 320% of the ground controls. Signals via G protein-coupled receptors are transmitted through PLC-beta and Ras GRF (another Ras GEF). Activated Ras then stimulates Raf, mitogen-activated protein kinase (MAPK) cascades. The mRNA levels of Raf, extracellular signal-regulated protein kinase of MAPK family (ERK-1 and -2), and PLC-beta were increased during spaceflight. Rho GAP expression in the flight cultures was increased twofold of the ground controls. Since Rho GAP deactivates Rho, microgravity may suppress Rho signals, regulating actin filament rearrangement. Microgravity signals may involve two pathways (G protein-coupled receptor-mediated pathway and tyrosine phosphorylation-mediated pathway) that activate Ras, Raf, and MAPK cascades in rat osteoblasts.
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PMID:Small GTPase Ras and Rho expression in rat osteoblasts during spaceflight. 1740 41

In fat and muscle cells, insulin stimulates the movement to and fusion of intracellular vesicles containing GLUT4 with the plasma membrane, a process referred to as GLUT4 translocation. Previous studies have indicated that Akt [also known as PKB (protein kinase B)] phosphorylation of AS160, a GAP (GTPase-activating protein) for Rabs, is required for GLUT4 translocation. The results suggest that this phosphorylation suppresses the GAP activity and leads to the elevation of the GTP form of one or more Rabs required for GLUT4 translocation. Based on their presence in GLUT4 vesicles and activity as AS160 GAP substrates, Rabs 8A, 8B, 10 and 14 are candidate Rabs. Here, we provide further evidence that Rab10 participates in GLUT4 translocation in 3T3-L1 adipocytes. Among Rabs 8A, 8B, 10 and 14, only the knockdown of Rab10 inhibited GLUT4 translocation. In addition, we describe the subcellular distribution of Rab10 and estimate the fraction of Rab10 in the active GTP form in vivo. Approx. 5% of the total Rab10 was present in GLUT4 vesicles isolated from the low-density microsomes. In both the basal and the insulin state, 90% of the total Rab10 was in the inactive GDP state. Thus, if insulin increases the GTP form of Rab10, the increase is limited to a small portion of the total Rab10. Finally, we report that the Rab10 mutant considered to be constitutively active (Rab10 Q68L) is a substrate for the AS160 GAP domain and, hence, cannot be used to deduce rigorously the function of Rab10 in its GTP form.
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PMID:Rab10 in insulin-stimulated GLUT4 translocation. 1807 83

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.
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PMID:Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes. 1821 34

The transcription factor Egr-1 is encoded by an immediate early response gene and has been shown to be a key regulator in the induction of apoptosis, mitogenesis and differentiation. It is rapidly induced by different stimuli including the glycoprotein hormone erythropoietin. In this report, we analyse the role of different erythropoietin receptor substructures for the activation of Egr-1 and the functional consequences of Egr-1 overexpression in the erythroleukemic cell line ELM-I-1. The investigation of receptor variants revealed that the activity of JAK2 and the phosphorylation of receptor tyrosine residues are essential preconditions for the ability to target Egr-1. Furthermore, we observed a close correlation of the abilities of receptors to activate the Ras-MAPK pathway and Egr-1. Using mass spectrometry we identified the Ras-GTPase-activating protein-SH3-domain-binding protein 1 (G3BP-1), a component of the Ras network of proteins, as an Egr-1 interacting protein in EPO stimulated ELM-I-1 cells. The overexpression of Egr-1 in these cells resulted in an enhanced rate of spontaneous erythroid differentiation.
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PMID:Erythropoietin receptor-mediated Egr-1 activation: structural requirements and functional implications. 1862 90

Ras-associated protein 1 (Rap1), a small GTPase, attracted attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes and of Rap1 effectors for angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVECs) express Rap1a and Rap1b mRNA. To determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase-activating protein that inhibits Rap1 activity. Overexpression of Rap1GAP1 significantly blocked angiogenic sprouting and tube-forming activity of HUVECs as well as migration and integrin-dependent adhesion. Silencing of Rap1a, Rap1b, or both significantly blocked HUVECs sprouting under basal and basic fibroblast growth factor-stimulated conditions and reduced HUVEC migration and integrin-dependent adhesion. We found that Rap1a and Rap1b are essential for the conformational activation of beta(1)-integrins in endothelial cells. Furthermore, silencing of Rap1a and Rap1b prevented phosphorylation of tyrosine 397 in focal adhesion kinase (FAK) and vascular endothelial growth factor-induced Akt1-activation. Rap1a(-/-)-deficient and Rap1a(+/-) heterozygote mice displayed reduced neovascularization after hind limb ischemia compared with wild-type mice. Silencing of RAPL significantly blocked the Rap1-induced sprouting of HUVECs, suggesting that the angiogenic activity of Rap1 is partly mediated by RAPL. Our data demonstrate a critical role of Rap1 in the regulation of beta(1)-integrin affinity, adhesion, and migration in endothelial cells and in postnatal neovascularization.
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PMID:Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis. 1880 68

The repulsive guidance molecule RGMa performs several functions in the developing and adult CNSs. RGMa, through its receptor neogenin, induces growth cone collapse and neurite outgrowth inhibition. Here, we demonstrate that RGMa binding to neogenin leads to the inactivation of Ras, which is required for the RGMa-mediated repulsive function in cortical neurons. This signal transduction is mediated by the Ras-specific GTPase-activating protein (GAP) p120GAP. The SH2 domain of p120GAP interacts with focal adhesion kinase (FAK), which is phosphorylated at Tyr-397. When the cells are stimulated with RGMa, FAK undergoes dephosphorylation at Tyr-397 and is dissociated from p120GAP, and this dissociation is followed by an increase in the interaction between p120GAP and GTP-Ras. In addition, the knockdown of p120GAP prevents RGMa-induced growth cone collapse and neurite outgrowth inhibition. Furthermore, RGMa stimulation induces Akt inactivation through p120GAP, and the expression of the constitutively active Akt prevents RGMa-induced growth cone collapse. Thus, RGMa binding to neogenin regulates p120GAP activity through FAK Tyr-397 dephosphorylation, leading to the inactivation of Ras and its downstream effector Akt, and this signal transduction plays a role in the RGMa-mediated repulsive function.
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PMID:Inactivation of Ras by p120GAP via focal adhesion kinase dephosphorylation mediates RGMa-induced growth cone collapse. 1945 35

Deleted in Liver Cancer 1 (DLC1) is a RHO GTPase-activating protein (GAP) that negatively regulates RHO. Through its GAP activity, it modulates the actin cytoskeleton network and focal adhesion dynamics, ultimately leading to suppression of cell invasion and metastasis. Despite its presence in various structural and signaling components, little is known about how the activity of DLC1 is regulated at focal adhesions. Here we show that EGF stimulation activates the GAP activity of DLC1 through a concerted mechanism involving DLC1 phosphorylation by MEK/ERK and its subsequent dephosphorylation by protein phosphatase 2A (PP2A) and inhibition of focal adhesion kinase by MEK/ERK to allow the binding between DLC1 and PP2A. Phosphoproteomics and mutation studies revealed that threonine 301 and serine 308 on DLC1, known previously to be mutated in certain cancers, are required for DLC1-PP2A interaction and the subsequent activation of DLC1 upon their dephosphorylation. The intricate interplay of this "MEK/ERK-focal adhesion kinase-DLC1-PP2A" quartet provides a novel checkpoint in the spatiotemporal control of cell spreading and cell motility.
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PMID:Epidermal growth factor activates the Rho GTPase-activating protein (GAP) Deleted in Liver Cancer 1 via focal adhesion kinase and protein phosphatase 2A. 2552 71

Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser(858) of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease.
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PMID:Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders. 2649 1

Male germ cell Rac GTPase-activating protein (MgcRacGAP) is a core regulator of cytokinesis. Furthermore, it appears to be involved in human oncogenesis through cytokinesis-independent mechanisms and has been reported to be essential for nuclear translocation of signal transducer and activator of transcription (STAT) proteins, including the oncoprotein STAT3. Here we utilized MgcRacGAP inhibitor compound 1 (MINC1), a small molecule inhibitor of MgcRacGAP, to further investigate how MgcRacGAP regulates STAT3. Surprisingly, both MINC1 treatment and small interference RNA (siRNA)-mediated gene silencing of MgcRacGAP resulted in increased STAT3 phosphorylation and STAT3-driven transcriptional activity in our experimental systems. Finally, we demonstrated that MINC1-induced STAT3 activation likely is due to increased STAT3 phosphorylation caused by a Rac1-PAR3-IL6-IL6R-JAK2 mediated autocrine/paracrine mechanism.
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PMID:MgcRacGAP inhibition stimulates JAK-dependent STAT3 activity. 2660 80

The receptor deleted in colorectal cancer (DCC) mediates the attraction of growing axons to netrin-1 during brain development. In response to netrin-1 stimulation, DCC becomes a signaling platform to recruit proteins that promote axon outgrowth and guidance. The Ras GTPase-activating protein (GAP) p120RasGAP inhibits Ras activity and mediates neurite retraction and growth cone collapse in response to repulsive guidance cues. Here we show an interaction between p120RasGAP and DCC that positively regulates netrin-1-mediated axon outgrowth and guidance in embryonic cortical neurons. In response to netrin-1, p120RasGAP is recruited to DCC in growth cones and forms a multiprotein complex with focal adhesion kinase and ERK. We found that Ras/ERK activities are elevated aberrantly in p120RasGAP-deficient neurons. Moreover, the expression of p120RasGAP Src homology 2 (SH2)-SH3-SH2 domains, which interact with the C-terminal tail of DCC, is sufficient to restore netrin-1-dependent axon outgrowth in p120RasGAP-deficient neurons. We provide a novel mechanism that exploits the scaffolding properties of the N terminus of p120RasGAP to tightly regulate netrin-1/DCC-dependent axon outgrowth and guidance.
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PMID:p120RasGAP Protein Mediates Netrin-1 Protein-induced Cortical Axon Outgrowth and Guidance. 2671 Aug 49


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