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)

STAT (signal transducers and activators of transcription) proteins are dual function proteins, which participate in cytokine-mediated signal transduction events at the cell surface and transcriptional regulation in the nucleus. We have exploited insights into the activation mechanism of STAT factors to derive constitutively active variants. Chimeric genes encoding fusion proteins of STAT5 and the kinase domain of JAK2 have been derived. The functional properties of the fusion proteins have been investigated in transiently transfected COS cells or in HeLa cells stably transfected with STAT5-JAK2 gene constructs regulated by a tetracycline-sensitive promoter. The STAT5-JAK2 proteins exhibit tyrosine kinase activity and are phosphorylated on tyrosine. The molecules are activated through an intramolecular or a cross-phosphorylation reaction and exhibit constitutive, STAT5-specific DNA binding activity. The transactivation potentials of three constitutively activated STAT5-JAK2 variants comprising different transactivation domains (TADs) derived from STAT5, STAT6, and VP16 were compared. The chimeric molecule containing the STAT5 TAD had no or only a very low, the molecule with the STAT6 TAD a medium, and the molecule with the VP16 TAD a very high transactivation potential. Transcription from STAT5-responsive gene promoter regions of the beta-casein, oncostatin M, and the cytokine-inducible Src homology 2 domain-containing protein genes was observed. These chimeric STAT molecules allow the study of the function of STAT5 independent of cytokine receptors and the activation of other signal transduction pathways.
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PMID:Cytokine receptor-independent, constitutively active variants of STAT5. 937 8

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. Binding of GM-CSF activates at least one receptor-associated tyrosine kinase, JAK2, and rapidly induces tyrosine phosphorylation of the GMR betac-chain (GMRbeta), but not the GMR alpha-chain (GMRalpha). To examine the role of GMRbeta tyrosine phosphorylaiton, each of the 8 tyrosine residues in the cytoplasmic domain of the human GMRbeta was mutated to phenylalanine (GMRbeta-F8), and this mutant receptor was expressed with wild-type GMRalpha in the interleukin-3-dependent murine hematopoietic cell line, Ba/F3. GM-CSF induced tyrosine phosphorylation of multiple cellular proteins in cells expressing GMRbeta-F8 , including JAK2 and STAT5. However, GM-CSF-induced tyrosine phosphorylation of both SHP2 and SHC was reduced or absent compared with wild-type. Next, a series of 8 receptors were generated, each containing only a single, restored, tyrosine residue. Tyrosine 577 was found to be sufficient to regenerate GM-CSF-dependent phosphorylation of SHC, and any of Y577, Y612, or Y695 was sufficient to regenerate GM-CSF-inducible phosphorylation of SHP2. Despite the signaling defect to SHC and SHP2, Ba/F3 cells expressing GMRbeta-F8 were still able to proliferate in response to 10 ng/mL of human GM-CSF, although mitogenesis was impaired compared with wild-type GMRbeta, and this effect was even more prominent at lower concentrations of GM-CSF (1 ng/mL). Overall, these results indicate that GMRbeta tyrosine residues are not necessary for activation of the JAK/STAT pathway or for proliferation, viability, or adhesion signaling in Ba/F3 cells, although tyrosine residues significantly affect the magnitude of the response. However, specific tyrosine residues are needed for activation of SHC and SHP2.
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PMID:Signaling functions of the tyrosine residues in the betac chain of the granulocyte-macrophage colony-stimulating factor receptor. 938 92

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

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

Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates many of the biological functions of human neutrophils. This includes the stimulation of protein synthesis and the tyrosine phosphorylation of various proteins among which is JAK2. The present study was aimed at characterizing in detail the pattern of activation by GM-CSF of the JAK/STAT pathway in human neutrophils. The results obtained show that the stimulation of human neutrophils by GM-CSF specifically led to tyrosine phosphorylation of JAK2 and had no effect on JAK1, JAK3, or TYK2. Furthermore, GM-CSF induced the tyrosine phosphorylation of STAT3 and STAT5 but not of STAT1, STAT2, STAT4, or STAT6. Tyrosine phosphorylation of STAT3 was transient reaching its maximum at 15 min. STAT5 presented a different pattern of tyrosine phosphorylation. The anti-STAT5 antibodies identified two proteins at 94 and 92 kDa. The 94-kDa STAT5 was constitutively tyrosine phosphorylated and showed no change upon GM-CSF stimulation. On the other hand, the 92-kDa STAT5 was tyrosine phosphorylated within 1 min of GM-CSF treatment and this was maintained for at least 30 min. By the use of specific antibodies, it was determined that only STAT5B, and not STAT5A, was tyrosine phosphorylated in GM-CSF-treated neutrophils. Furthermore, GM-CSF treatment induced an increase in the ability of STAT3 and STAT5B, but not STAT5A, to bind DNA probes. The specificity of the pattern of activation of the JAK/STAT pathway suggests that it may be directly linked to the modulation of the functions of mature nondividing, human neutrophils by GM-CSF.
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PMID:Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. Selective activation of Jak2, Stat3, and Stat5b. 942 69

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

The G401 cell line derived from a rhabdoid tumor of the kidney secretes the heparin-binding growth factors midkine and pleiotrophin. Both proteins act as mitogens for diverse cells, but only midkine serves as an autocrine mitogen for G401 tumor cells. We show that midkine specifically binds a protein or complex of molecular mass greater than 200 kDa with high affinity (Kd = 0.07 +/- 0.01 nM). Midkine, but not pleiotrophin, stimulates tyrosine phosphorylation of several cellular proteins with molecular mass of 100, 130, and 200+ kDa. Upon midkine binding, the midkine-receptor complex associates with the Janus tyrosine kinases, JAK1 and JAK2. MK stimulates tyrosine phosphorylation of JAK1, JAK2, and STAT1alpha. Our initial characterization of the midkine receptor suggests that midkine autocrine stimulation of tumor cell proliferation is mediated by a cell-surface receptor which in turn might activate the JAK/STAT pathway.
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PMID:Midkine induces tumor cell proliferation and binds to a high affinity signaling receptor associated with JAK tyrosine kinases. 945 95

Interferons and cytokines modulate gene expression via a simple, direct signaling pathway containing receptors, JAK tyrosine kinases, and STAT transcription factors. The interferon-alpha pathway is a model for these cascades. Two receptors, IFNaR1 and IFNaR2, associate exclusively in a constitutive manner with two JAK proteins, TYK2 and JAK1, respectively. Defining the molecular interface between JAK proteins and their receptors is critical to understanding the signaling pathway and may contribute to the development of novel therapeutics. This report defines the IFNaR1 interaction domain on TYK2. In vitro binding studies demonstrate that the amino-terminal half of TYK2, which is approximately 600 amino acids long and contains JAK homology (JH) domains 3-7, comprises the maximal binding domain for IFNaR1. A fragment containing amino acids 171-601 (JH3-6) also binds IFNaR1, but with reduced affinity. Glutathione S-transferase-TYK2 fusion proteins approximating either the JH6 or JH3 domain affinity-precipitate IFNaR1, suggesting that these are major sites of interaction within the larger binding domain. TYK2 amino acids 1-601 act in a dominant manner to inhibit the transcription of an interferon-alpha-dependent reporter gene, presumably by displacing endogenous TYK2 from the receptor. This same fragment inhibits interferon-alpha-dependent tyrosine phosphorylation of TYK2, STAT1, and STAT2.
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PMID:Definition of the interferon-alpha receptor-binding domain on the TYK2 kinase. 946 96

This study was designed to demonstrate the characteristic pattern of angiotensin II-induced JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) activation in cultured rat cardiomyocytes by comparing it with leukemia inhibitory factor (LIF)-induced activation. Angiotensin II (10(-7) mol/L) induced rapid phosphorylation of JAK2 and Tyk2, but not JAK1, and phosphorylated STAT1 and STAT2, but not STAT3, in the early stage up to 30 minutes. The time course of JAK/STAT activation by angiotensin II was apparently slower than that by LIF. Interestingly, angiotensin II phosphorylated STAT3 and rephosphorylated STAT1 in the late stage at 120 minutes. We also found that angiotensin II induced the formation of interferon-stimulating gene factor (ISGF) complexes biphasically, in the early stage at 15 to 30 minutes and in the late stage at 120 minutes, and that angiotensin II induced delayed activation of the sis-inducing factor (SIF) complex at 120 minutes. Formation of ISGF and SIF complexes in response to angiotensin II paralleled the phosphorylation pattern of STAT1 and STAT3 and was quite different from those obtained in response to LIF. The phosphorylation of STAT1 was suppressed by pretreatment with the angiotensin II type-1 (AT1) receptor antagonist CV11974, but the delayed addition of CV11974 failed to suppress phosphorylation of STAT3 at 120 minutes. In conclusion, angiotensin II-induced JAK/STAT activation in rat cardiomyocytes is biphasic and entirely different from LIF-induced activation.
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PMID:Biphasic activation of the JAK/STAT pathway by angiotensin II in rat cardiomyocytes. 946 95

The interleukin-3 dependent murine Ba/F3 cell line has been widely used as an experimental model of cell transformation by BCR-ABL oncogenes as assessed by induction of growth-factor-independence and inhibition of apoptosis in vitro. The signaling pathways used by BCR-ABL oncogenes to exert these effects are unknown. To gain insights into this phenomenon, we have introduced the p190- and p210-encoding BCR-ABL oncogenes as well as the constitutively activated oncogenic murine erythropoietin receptor (cEpoR) into Ba/F3 and compared the behavior of individual clones in response to apoptotic stimuli. Both p210 and p190 BCR-ABL vectors induced IL-3-independent growth and the same result was obtained with the cEpo-R vector. Individual clones of Ba/F3 cells expressing BCR-ABL exhibited significant resistance to apoptosis induced by either etoposide, serum deprivation or growth-factor withdrawal. In contrast, Ba/F3 cells expressing the constitutively active cEpoR behaved like parental Ba/F3 cells undergoing apoptosis when similarly treated with etoposide or upon serum deprivation. Bc12 and Bax levels were similar in all BCR-ABL and cEpoR-transfected clones. However, in band-shift assays, nuclear extracts from growth-factor-independent Ba/F3 clones expressing cEpoR had no detectable STAT activity as opposed to the constitutive STAT activation detected in all Ba/F3 clones expressing p210 or p190 BCR-ABL. Our results indicate that although both constitutively activated cEpoR and BCR-ABL oncogenes induce growth-factor independence in Ba/F3 cells, only BCR-ABL is able to protect cells from etoposide and serum-deprivation-induced apoptosis and induce a strong constitutive activation of STAT factors, suggesting a role for these molecules in the anti-apoptotic activity of BCR-ABL.
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PMID:BCR-ABL and constitutively active erythropoietin receptor (cEpoR) activate distinct mechanisms for growth factor-independence and inhibition of apoptosis in Ba/F3 cell line. 948 38


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