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)

Growth hormone (GH) treatment of cells promotes activation of JAK2, a GH receptor (GHR)-associated tyrosine kinase. We now explore JAK2 regions required for GHR-induced signaling. Wild-type (WT) JAK2 and JAK2 molecules with deletions of the amino terminus (JAK2ATD), carboxyl terminus (JAK2CTD), or kinase-like domain (JAK2PKD) were each transiently coexpressed in COS-7 cells with the rabbit GHR. The following responses were assayed: GH-induced transactivation of a luciferase reporter governed by a c-fos enhancer element; GH-induced shift in the molecular mass of a cotransfected epitope-tagged extracellular signal-regulated kinase molecule; and GH-induced antiphosphotyrosine immunoprecipitability of the transfected JAK2 form. In each assay, WTJAK2 and JAK2PKD allowed GH-induced signaling, whereas JAK2ATD and JAK2CTD did not. Anti-GHR serum coimmunoprecipitated WTJAK2, JAK2PKD, and JAK2CTD, but not JAK2ATD. Finally, a chimera in which the JAK2 kinase domain replaced the GHR cytoplasmic domain signaled GH-induced transactivation. We conclude: 1) kinase-like domain deletion eliminates neither physical nor functional interaction between JAK2 and the GHR; 2) kinase domain deletion eliminates functional but not physical coupling of JAK2 to the GHR; 3) interaction with the GHR appears dependent on the NH2-terminal one-fifth of JAK2; and 4) a GH-responsive signaling unit can include as little as the GHR external and transmembrane domains and the JAK2 kinase domain.
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PMID:Regions of the JAK2 tyrosine kinase required for coupling to the growth hormone receptor. 754 Jan 78

The proliferation of activated T lymphocytes is critically dependent on the binding of the T-cell growth factors, interleukin (IL)-2 and IL-4, to distinct but evolutionarily related cell surface receptors. Previous results suggest that the IL-2 receptor (IL-2R) and IL-4R are coupled to both overlapping and distinct intracellular signaling pathways in T lymphocytes. In this study, we demonstrate that activation of Janus tyrosine kinases (JAKs) and STAT transcription factors is rapidly induced by exposure of factor-dependent murine T-cell lines to IL-2 or IL-4. Both IL-2 and IL-4 stimulated the rapid activation of JAK1 and JAK3, whereas JAK2 activity was unaffected by either cytokine. These responses were accompanied by the appearance in cell nuclei of 3 DNA binding activities that recognized a high-affinity binding site for STAT factors. In transient transfection assays, this STAT factor target sequence conferred IL-2 and IL-4 inducibility on a synthetic luciferase reporter gene. Antibody supershifting experiments indicated that IL-2 induces the formation of STAT dimers containing STAT3 and STAT1 alpha. Although IL-4 also activated STAT1 alpha, the major IL4-induced STAT factor is not STAT3 and remains undefined. Pretreatment of the T-cells with the protein-tyrosine kinase inhibitor herbimycin A blocked both the nuclear translocation of STAT factors and STAT-dependent reporter gene transcription. Immunoblot analyses confirmed that cytoplasmic STAT3 was heavily phosphorylated on tyrosine in IL-2-stimulated cells, and that phosphorylated STAT3 appeared in the nuclei of these cells. These results indicate that identical JAKs and partially overlapping sets of STATs are activated by IL-2 and IL-4 in T lymphocytes.
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PMID:Protein-tyrosine kinase-dependent activation of STAT transcription factors in interleukin-2- or interleukin-4-stimulated T lymphocytes. 774 3

The gene defective in X-linked agammaglobulinemia (XLA) encodes a novel protein kinase termed Bruton's tyrosine kinase (Btk). Whereas the XLA phenotype is confined to abnormalities of B-cell development and function, Btk is expressed not only in B-lymphocyte lineage but also in myeloid lineage cells. The first 450 basepairs of the Btk promoter fused to a luciferase gene displayed a similar cell-type specificity. Critical binding sites for the transcription factors PU.1 and Sp1 were identified in the proximal portion of the Btk promoter upstream of a cluster of transcriptional start sites. Mutation of either the PU.1 or Sp1 site markedly reduced the activity of a Btk promoter-luciferase reporter construct in transfection experiments. In addition, PU.1 directly transactivated the Btk promoter, and deletion of the PU.1 binding site abolished this effect. This study implicates PU.1 and Sp1 as major regulators of Btk expression and provides a foundation for further study of the regulation of this gene in XLA patients that lack Btk mRNA.
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PMID:Analysis of the Bruton's tyrosine kinase gene promoter reveals critical PU.1 and SP1 sites. 856 28

Cytokine receptors activate multiple signal transduction pathways, resulting in the induction of specific target genes. We have recently identified a hematopoietic cell-specific immediate-early gene, DUB-1, that encodes a growth-regulatory deubiquitinating enzyme. The DUB-1 gene contains a 112-bp enhancer element that is specifically induced by the beta c subunit of the interleukin-3 (IL-3) receptor. To investigate the mechanism of DUB-1 induction, we examined the effects of dominant-negative forms of JAK kinases, STAT transcription factors, and Raf-1 in transient transfection assays. In Ba/F3 cells, IL-3 induced a dose-dependent activation of DUB-1-luciferase (luc) and GAS-luc reporter constructs. A dominant-negative form of JAK2 (truncated at amino acid 829) inhibited the induction of DUB-1-luc and GAS-luc by IL-3. A dominant-negative form of STAT5 (truncated at amino acid 650) inhibited the induction of GAS-luc but not DUB-1-luc. A dominant-negative form of Raf-1 inhibited the induction of DUB-1-luc but had no effect on the induction of GAS-luc by IL-3. The requirement for JAK2 in the stimulation of the DUB-1 enhancer was further supported by the suppression of DUB-1 induction in Ba/F3 cells stably expressing the dominant-negative JAK2 polypeptide. We hypothesize that IL-3 activates a JAK2/Raf-1 signaling pathway that is required for DUB-1 induction and is independent of STAT5.
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PMID:JAK2 is required for induction of the murine DUB-1 gene. 915 35

Interleukin-6 (IL-6) is an important B-cell growth and differentiation factor. IL-6 treatment of the human lymphoblastoid cell line, SKW6.4, leads to increased IgM production. We have previously shown that IL-6 induces activation of JAK1 and JAK2 in human B cell lines. A chimeric IL-6 receptor, comprised of the intracellular tail of the IL-6 receptor subunit gp130 fused to the extracellular domain of the epidermal growth factor (EGF) receptor, was stably transfected into SKW6.4 cells. EGF treatment induced IgM production in cells transfected with an intact gp130 cytoplasmic tail, but not in untransfected cells or cells transfected with a cytoplasmic tail lacking all four signal transducers and activators of transcription (Stat) binding sites. Moreover, EGF treatment induced Stat3 phosphorylation in cells transfected with the intact chimeric EGF-gp130 receptor along with induction of DNA-mobility shift of a classical interferon-gamma-activated site. To define further the relation between Stat3 activation and enhanced IgM production, we determined the effect of chimeric gp130 on the transcriptional activation of a genetic element linked to immunoglobulin production, namely the immunoglobulin heavy chain enhancer (IgH-enhancer). Parental as well as transfected SKW6.4 cells were transiently transfected with an IgH-enhancer-luciferase construct. The transcriptional activity of the IgH-luciferase construct was induced upon ligation of the full-length chimeric receptor but not by truncated gp130 receptors. Moreover, the gp130-induced activity of this reporter gene was abrogated by Stat3EE, a mutant Stat3 incapable of binding DNA. These results indicate that IL-6-induced B-cell differentiation, as measured by IgM production, may be controlled by Stat3 proteins.
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PMID:Involvement of Stat3 in interleukin-6-induced IgM production in a human B-cell line. 915 40

Leukemia inhibitory factor (LIF) is a cytokine that plays an important role during mouse embryogenesis. We showed that adenovirus E1A represses the interleukin-6 signal transduction pathway that uses the same JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor as LIF. Here, we report that the LIF-JAK-STAT signal transduction pathway is blocked in cellular E1A-expressing undifferentiated F9 cells, and that the block is overcome by retinoic acid-induced differentiation. LIF failed to stimulate the expression of the acute phase response element (APRE)-driven luciferase gene in undifferentiated F9 cells, whereas the luciferase activity was remarkably increased by LIF treatment in differentiated F9 (dF9) cells. We analyzed the mechanism of the APRE regulation and found that the LIF-induced APRE-binding activity was regulated in a differentiation-dependent manner. The protein levels and the tyrosine phosphorylation of JAK1, JAK2, and STAT3 in F9 cells were not different from those in dF9 cells. The exogenous expression of activated c-Ha-ras partially recovered the LIF responsiveness of the APRE-luciferase gene in F9 cells, but the dominant negative ras N-17 did not repress the LIF-induced activation of APRE-luciferase in dF9 cells. These results suggested that an unknown coactivation process that is partially compensated by Ras is required for STAT3-APRE binding in F9 cells.
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PMID:Alternative signaling mechanism of leukemia inhibitory factor responsiveness in a differentiating embryonal carcinoma cell. 920 5

The interaction of prolactin (PRL) with its receptor leads to activation of the tyrosine kinase, Janus kinase 2 (JAK2). In the cytoplasmic juxtamembrane region, a short segment (Box 1) which is conserved in other receptors of the PRL/growth hormone (GH)/cytokine receptor family, is required for signal transduction. To assess the contribution of the different amino acids of Box 1, individual alanine substitutions of all residues, grouped substitution of four prolines (4PA mutant) and individual leucine replacement of the two last prolines (P248L and P250L mutants) were introduced. Here we show that P250L and 4PA (i) inhibit PRL-induced transactivation of a luciferase reporter governed by a beta-caseine gene promoter; (ii) decrease in JAK2 tyrosine kinase activity in biotinylated-PRL precipitates; (iii) impair the interaction between PRLR and JAK2, as evidenced by lack of co-immunoprecipitation, (iv) and prevent the activation of signal transducer and activator of transcription (Stat) as determined by absence of tyrosine phosphorylation of Stat5. Our data suggest that the Box 1 region of the PRL receptor and particularly the last proline is critical for JAK2 association and subsequent activation. These results support the notion that the tyrosine kinase JAK2 is implicated in activation of downstream protein effectors such as Stat5, which are involved in transcription of PRL-responsive genes.
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PMID:The last proline of Box 1 is essential for association with JAK2 and functional activation of the prolactin receptor. 920 3

In addition to a long form of 591 amino acids (aa), two other forms of PRL receptor (PRLR), differing in the length of their cytoplasmic domains, have been identified in the rat. The Nb2 form, lacking 198 aa in the cytoplasmic domain, is able to transmit a lactogenic signal similar to the long form, whereas the short form of 291 aa is inactive. The ability of PRL to activate the promoter of the beta-casein gene or the lactogenic hormone responsive element fused to the luciferase reporter was assessed in Chinese hamster ovary cells or 293 fibroblasts transiently transfected with PRLR cDNAs. The function of the short form was examined after cotransfection of both the long and short forms. These results clearly show that the short form acts as a dominant negative inhibitor through the formation of inactive heterodimers, resulting in an inhibition of Janus kinase 2 (JAK2) activation. The present study also investigates the possible participation of cytoplasmic receptors in the signal transduction pathway, using cotransfection experiments and a new approach that selectively determines the contribution of cytoplasmic receptors in the process of signal transduction. We cotransfected Chinese hamster ovary cells with two cDNA constructs: a cytoplasmic (soluble) form of the receptor with a deleted signal peptide (delta-19), which is unable to bind PRL, and a functionally inactive receptor mutant (lacking box 1), which is anchored in the plasma membrane and able to bind PRL. This approach has allowed us to show that delta-19, lacking expression at the plasma membrane, can transduce the hormonal message, at least to a limited extent (up to 30% of wild type efficiency), providing that association/activation occurs with a PRL-PRLR complex initiated at the cell surface level; box 1 of the cytoplasmic form is necessary to rescue this partial transcriptional activity of the inactive mutant. This partial recovery is also parallel to the partial activation of JAK2, indicating that the signal transduction pathway implicated JAK2. Our results provide evidence that heterodimerization of receptors can be implicated either in the positive or in negative activation of gene transcription.
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PMID:Dominant negative and cooperative effects of mutant forms of prolactin receptor. 921 50

Leptin receptors include a long form (OBRl) with 302 cytoplasmic residues that is presumed to mediate most or all of leptins signaling, and several short forms, including one (OBRs) that has 34 cytoplasmic residues, is widely expressed, and is presumed not to signal but to mediate transport or clearance of leptin. We studied the abilities of these two receptor isoforms to mediate signaling in transfected cells. In response to leptin, OBRl, but not OBRs, underwent tyrosine phosphorylation that was enhanced by co-expression with JAK2. In cells expressing receptors and JAK2, both OBRs and OBRl mediated leptin-dependent tyrosine phosphorylation of JAK2, and this was abolished with OBRs when the Box 1 motif was mutated. In cells expressing receptors, JAK2 and IRS-1, leptin induced tyrosine phosphorylation of IRS-1 through OBRs and OBRl. In COS cells expressing hemagglutinin-ERK1 and receptors, leptin increased ERK1 kinase activity through OBRl, with the magnitude increased by co-expression of JAK1 or JAK2, and to a lesser degree through OBRs, despite greater receptor expression. In stable Chinese hamster ovary cell lines expressing OBRs or OBRl, leptin stimulated endogenous ERK2 phosphorylation. Whereas leptin stimulated tyrosine phosphorylation of hemagglutinin-STAT3 and induction of a c-fos luciferase reporter plasmid through OBRl, OBRs was without effect in these assays. In conclusion, OBRl is capable of signaling to IRS-1 and mitogen-activated protein kinase via JAK, in addition to activating STAT pathways. Although substantially weaker than OBRl, OBRs is capable of mediating signal transduction via JAK, but these activities are of as yet unknown significance for leptin biology in vivo.
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PMID:Divergent signaling capacities of the long and short isoforms of the leptin receptor. 940 87

Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-alpha-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485-620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.
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PMID:Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling. 944 80


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