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)

The cytokine-inducible SH2 protein-3 (CIS3/SOCS-3/SSI-3) has been shown to inhibit the JAK/STAT pathway and act as a negative regulator of fetal liver erythropoiesis. Here, we studied the molecular mechanisms by which CIS3 regulates the erythropoietin (EPO) receptor (EPOR) signaling in erythroid progenitors and Ba/F3 cells expressing the EPOR (BF-ER). CIS3 binds directly to the EPOR as well as JAK2 and inhibits EPO-dependent proliferation and STAT5 activation. We have identified the region containing Tyr(401) in the cytoplasmic domain of the EPOR as a direct binding site for CIS3. Deletion of the Tyr(401) region of the EPOR reduced the inhibitory effect of CIS3, suggesting that binding of CIS3 to the EPOR augmented the negative effect of CIS3. Both N- and C-terminal regions adjacent to the SH2 domain of CIS3 were necessary for binding to EPOR and JAK2. In the N-terminal region of CIS3, the amino acid Gly(45) was critical for binding to the EPOR but not to JAK2, while Leu(22) was critical for binding to JAK2. The mutation of G45A partially reduced ability of CIS3 to inhibit EPO-dependent proliferation and STAT5 activation, while L22D mutant CIS3 was completely unable to suppress EPOR signaling. Moreover, overexpression of STAT5, which also binds to Tyr(401), reduced the binding of CIS3 to the EPOR, and the inhibitory effect of CIS3 against EPO signaling, while it did not affect JAB/SOCS-1/SSI-1. These data demonstrate that binding of CIS3 to the EPOR augments the inhibitory effect of CIS3. CIS3 binding to both EPOR and JAK2 may explain a specific regulatory role of CIS3 in erythropoiesis.
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PMID:CIS3/SOCS-3 suppresses erythropoietin (EPO) signaling by binding the EPO receptor and JAK2. 1088 25

Thrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit (amino acid residues 373-740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. Patients with hemophilia A have been described whose plasmas display a discrepancy between their FVIII activities, where the 1-stage activity assay displays greater activity than the 2-stage activity assay. The molecular basis for one of these mutations, (ARG)531(HIS), is an increased rate of A2 subunit dissociation. Examination of a homology model of the A domains of FVIII predicted (ARG)531 to lie at the interface of the A1 and A2 subunits and stabilize their interaction. Indeed, patients with mutations either directly contacting (ARG)531 ((ALA)284(GLU), (ALA)284(PRO)) or closely adjacent to the A1-A2 interface in the tightly packed hydrophobic core ((SER)289(LEU)) have the same phenotype of 1-stage/2-stage discrepancy. The (ALA)284(GLU) and (SER)289(LEU) mutations in FVIII were produced by transfection of COS-1 monkey cells. Compared to FVIII wild-type both mutants had reduced specific activity by 1-stage clotting activity and at least a 2-fold lower activity by 2-stage analysis (COAMATIC), similar to the reported clinical data. Analysis of immunoaffinity purified (ALA)284(GLU) and (SER)289(LEU) proteins in an optical biosensor demonstrated that A2 dissociation was 3-fold faster for both FVIIIa mutants compared to FVIIIa wild-type. Therefore, these mutations within the A1 subunit of FVIIIa introduce a similar destabilization of the FVIIIa heterotrimer compared to the (ARG)531(HIS) mutation within the A2 subunit and support that these residues stabilize the A domain interface of FVIIIa.
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PMID:Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa. 1115 85

The cellular and molecular basis of growth hormone (GH) actions on the heart remain poorly defined, and it is unclear whether GH effects on the myocardium are direct or mediated at least in part via insulin-like growth factor (IGF-1). Here, we demonstrate that the cultured neonatal cardiomyocyte is not an appropriate model to study the effects of GH because of artifactual loss of GH receptors (GHRs). To circumvent this problem, rat neonatal cardiomyocytes were infected with a recombinant adenovirus expressing the murine GHR. Functional integrity of GHR was suggested by GH-induced activation of the cognate JAK2/STAT5, MAPK, and Akt intracellular pathways in the cells expressing GHR. Although exposure to GH resulted in a significant increase in the size of the cardiomyocyte and increased expression of c-fos, myosin light chain 2, and skeletal alpha-actin mRNAs, there were no significant changes in IGF-1 or atrial natriuretic factor mRNA levels in response to GH stimulation. In this model, GH increased incorporation of leucine, uptake of palmitic acid, and abundance of fatty acid transport protein mRNA. In contrast, GH decreased uptake of 2-deoxy-d-glucose and levels of Glut1 protein. Thus, in isolated rat neonatal cardiomyocytes expressing GHR, GH induces hypertrophy and causes alterations in cellular metabolic profile in the absence of demonstrable changes in IGF-1 mRNA, suggesting that these effects may be independent of IGF-1.
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PMID:Demonstration of direct effects of growth hormone on neonatal cardiomyocytes. 1130 22

Tyrosine 1062 of Ret, which represents an intracytoplasmic docking site for multiple signaling molecules, is essential for Ret-mediated activation of phosphatidylinositol 3-Kinase (PI3-K). PI3-K, in turn, has been implicated in inducing cell survival and neoplastic transformation mediated by Ret. We have examined the mechanisms by which Ret stimulates PI3-K. Here we show that the Insulin Receptor Substrate-1 (IRS-1) is tyrosine phosphorylated and associated with the p85 regulatory subunit of PI3-K in response to Ret activation. IRS-1 coimmunoprecipitates with Ret and co-expression of IRS-1 results in the potentiation of Ret-mediated activation of Akt(PKB), a bona fide effector of PI3-K. The association with the PTB domain of IRS-1 depends on the phosphorylation of tyrosine 1062 of Ret. The deletion of asparagine 1059 (delN1059) and the substitution of leucine 1061 (L1061P), two Ret mutations identified in families affected by congenital megacolon (Hirschsprung's disease), impair the binding of IRS-1 to Ret as well as Ret-mediated Akt(PKB) stimulation. Finally, we show that Shc, which was previously identified as another ligand of Y1062 of Ret, competes with IRS-1 for the binding to Ret pY1062. All together, these findings suggest that IRS-1 is a component of the signaling pathway which leads to Ret-mediated PI3-K activation, a pathway which can be targeted by Hirschsprung-associated Ret mutations. The alternative binding of Shc and IRS-1 to Ret pY1062 can be a system to modulate the activation of different intracellular signaling pathways and to elicit different biological responses following Ret activation.
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PMID:The insulin receptor substrate (IRS)-1 recruits phosphatidylinositol 3-kinase to Ret: evidence for a competition between Shc and IRS-1 for the binding to Ret. 1131 48

In the absence of survival-inducing cytokines activated T cells and neutrophils enter apoptosis spontaneously. Phosphatidylinositol 3-kinase (PI3 K) activation and signaling through PKB/AKT have been widely linked to the inhibition of apoptosis by cytokines. Here we have investigated the role of PKB in the inhibition of spontaneous apoptosis of activated human CD4+ T cells and neutrophils. We used a range of cytokines known to induce survival and/or activation of PKB. We found activation of PKB in T cells treated with IL-2 and insulin, and neutrophils cultured with N-formyl-Met-Leu-Phe (fMLP), insulin or granulocyte-macrophage colony-stimulating factor. Insulin did not inhibit apoptosis in neutrophils or T cells and fMLP did not delay neutrophil apoptosis. Intriguingly, IFN-beta induced PI3 K-dependent survival in both cell types, but did not activate PKB. IL-2 mediated rescue of T cells from apoptosis but no induction of proliferation occurred in thepresence of LY294002, an inhibitor of PI3 K, which also blocked subsequent PKB activation. The main role of PI3 K in IL-2-mediated signaling may therefore be in the regulation of proliferation. These findings suggest that activation of PKB and inhibition of apoptosis can be dissociated in cytokine-mediated rescue of non-transformed CD4+ T cells and neutrophils.
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PMID:Cytokine-mediated inhibition of apoptosis in non-transformed T cells and neutrophils can be dissociated from protein kinase B activation. 1182 65

The thrombopoietin (TPO) receptor c-Mpl, like other members of the cytokine receptor superfamily, requires the association and activation of Janus kinases (JAKs) for normal signal transduction. The membrane-proximal portion of the signaling domain, containing conserved box1 and box2 motifs, is sufficient to support the proliferation of cytokine-dependent cell lines and basal megakaryocytopoiesis in vivo. We hypothesized that activation of the JAK2 kinase alone might be sufficient for proliferative signaling. To test this premise, we constructed chimeric receptors in which the extracellular and transmembrane portions of Mpl were fused to the pseudokinase and kinase domains of murine JAK2 kinase. When expressed in the interleukin-3-dependent cell line Ba/F3, the chimeric receptors were appropriately expressed on the cell surface and were able to initiate tyrosine kinase activity upon exposure to TPO. However, chimeric receptors lacking an intact box2 domain of Mpl were unable to support proliferation at any concentration of TPO. Only chimeric receptors containing both JAK2 kinase activity and the box2 region initiated proliferative signaling. Within the box2 motif, we determined that the sequence Glu(56)-Ile(57)-Leu(58) of the Mpl cytoplasmic domain is critical for proliferation of the chimeric receptors. Furthermore, TPO-dependent induction of c-myc transcription is also dependent on this motif. These results indicate that JAK2 activation alone is not sufficient for TPO-induced proliferation and that one or more essential signaling pathways must arise from the cytoplasmic domain of Mpl that includes box2. Although the nature of the signal transduction pathway is not yet known, this second proliferative event is likely to regulate c-myc expression.
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PMID:Studies with chimeric Mpl/JAK2 receptors indicate that both JAK2 and the membrane-proximal domain of Mpl are required for cellular proliferation. 1198 Sep 1

We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.
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PMID:Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. 1204 68

The transcriptional coactivator p/CIP is a member of a family of nuclear receptor coactivator/steroid receptor coactivator (NCoA/SRC) proteins that mediate the transcriptional activities of nuclear hormone receptors. We have found that p/CIP is predominantly cytoplasmic in a large proportion of cells in various tissues of the developing mouse and in a number of established cell lines. In mouse embryonic fibroblasts, serum deprivation results in the redistribution of p/CIP to the cytoplasmic compartment and stimulation with growth factors or tumor-promoting phorbol esters promotes p/CIP shuttling into the nucleus. Cytoplasmic accumulation of p/CIP is also cell cycle dependent, occurring predominantly during the S and late M phases. Leptomycin B (LMB) treatment results in a marked nuclear accumulation, suggesting that p/CIP undergoes dynamic nuclear export as well as import. We have identified a strong nuclear import signal in the N terminus of p/CIP and two leucine-rich motifs in the C terminus that resemble CRM-1-dependent nuclear export sequences. When fused to green fluorescent protein, the nuclear export sequence region is cytoplasmic and is retained in the nucleus in an LMB-dependent manner. Disruption of the leucine-rich motifs prevents cytoplasmic accumulation. Furthermore, we demonstrate that cytoplasmic p/CIP associates with tubulin and that an intact microtubule network is required for intracellular shuttling of p/CIP. Immunoaffinity purification of p/CIP from nuclear and cytosolic extracts revealed that only nuclear p/CIP complexes possess histone acetyltransferase activity. Collectively, these results suggest that cellular compartmentalization of NCoA/SRC proteins could potentially regulate nuclear hormone receptor-mediated events as well as integrating signals in response to different environmental cues.
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PMID:Microtubule-dependent subcellular redistribution of the transcriptional coactivator p/CIP. 1219 59

The insect neuropeptides FMRF amide, leucomyosupressin (LMS) and neuropeptide analogues leucosulfakinins (FLSK and LSK II Ser (SO(3)H)), perisulfakinin (PSK), proleucosulfakinin (PLSK), 14A[phi1]WP-I, 542phi1, and 378A[5b]WP-I were assayed for their effects on the release of amylase and protease from the midgut tissue of larvae of Opisina arenosella. In the bioassay, empty midgut tubes ligated at both ends using hair were incubated with insect saline containing neuropeptides/analogues in a bioassay apparatus at 37 degrees C for 30 min. After incubation the contents of the midgut preparations were analyzed for amylase and protease activity. In control experiments, the midgut preparations were incubated in insect saline without neuropeptides. The results of the study reveal that for stimulating amylase release from midgut tissue, the peptides require an FXRF amide (X may be methionine or leucine) sequence at the C-terminal. The presence of HMRF amide at C-terminal of peptides may inhibit the release of amylase. Meanwhile, peptides with both FMRF and HMRF amide sequence at the C-terminal are found to be effective in stimulating protease release. The tetrapeptide segment at the C-terminal probably represent the active core of the neuropeptide.
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PMID:In vitro release of digestive enzymes by FMRF amide related neuropeptides and analogues in the lepidopteran insect Opisina arenosella (Walk.). 1238 63

We characterized the overall rate of F-actin polymerization in the pseudopod region by measuring the rate of extension of single pseudopods stimulated by f-Met-Leu-Phe. The rate of pseudopod extension was measured in the presence of inhibitors for signaling molecules that are known to be involved in motility. Our data show the existence of 2 distinct signaling pathways of actin polymerization in the pseudopod region: a phosphoinositide 3-kinase gamma (PI3Kgamma)-dependent and -independent pathway. The PI3Kgamma dependent signaling of F-actin polymerization also depends on protein kinase C zeta and protein kinase B (Akt/PKB). The PI3Kgamma-independent pathway depends on GTPase RhoA, the RhoA ROCK kinase, Src family tyrosine kinases, and NADPH, and is modulated by cAMP.
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PMID:Chemoattractant receptor-stimulated F-actin polymerization in the human neutrophil is signaled by 2 distinct pathways. 1239 89


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