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)

To determine the specificity of signal transducer and activator of transcription (STAT) protein activation by box 3 motif-deficient hematopoietin receptors, expression vectors encoding the receptors for growth hormone, interleukin-3 (IL-3), and IL-4 were transiently transfected into COS-1 cells, together with expression vectors for Janus kinases (JAKs) and STAT proteins. Each receptor mediated a dose-dependent activation of STAT1 and STAT3, and for IL-3R and GHR this process was enhanced by JAK2. The data suggest that a box 3 motif in the cytoplasmic domain of the signal-transducing receptor to the JAK/STAT pathway. Transfection of the receptors, in combination with STAT3, into HepG2 cells reconstituted a cytokine-dependent stimulation of gene transcription through IL-6 response elements, providing evidence for a functional role of STAT3 in controlling gene expression.
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PMID:Receptors for interleukin-3 (IL-3) and growth hormone mediate an IL-6-type transcriptional induction in the presence of JAK2 or STAT3. 765 99

An early step in GH action involves tyrosine phosphorylation of various cellular proteins. Recently, it has been shown in murine preadipocytes that GH promotes the association of its receptor (the GHR) with and the activation of the JAK2 tyrosine kinase. In this study, we confirmed the human (h) GH-induced association of JAK2 with hGHR in IM-9 cells by coimmunoprecipitation experiments using anti-hGHR serum. We further examined the interaction of JAK2 with the GHR cytoplasmic domain by two lines of investigation. For in vitro studies, we assayed by immunoblotting the ability of cell-derived JAK2 to interact with glutathione S-transferase fusion proteins containing elements of the hGHR cytoplasmic domain. A fusion protein containing the entire hGHR cytoplasmic domain (residues 271-620) specifically associated with JAK2 independent of prior stimulation of cells with hGH. This interaction was not dependent on tyrosine phosphorylation of either partner. Mutational analysis of the hGHR cytoplasmic domain component of the fusions indicated that a membrane-proximal 20-residue region that includes the proline-rich box 1 was necessary for the interaction. This region appeared to cooperate with another region(s), largely in the N-terminal one third of the cytoplasmic domain, to promote full interaction with JAK2. For in vivo reconstitution experiments, wild-type (WT) and mutant rabbit GHRs (rGHRs) along with murine JAK2 were expressed by transient transfection in COS-7 cells. rGHR mutations were confined to the cytoplasmic domain and included C-terminal truncations as well as internal deletions of residues 297-406 and 278-292 (the latter contains box 1). All mutant rGHRs were expressed at the cell surface and bound hGH to a degree similar to the WT rGHR. Receptors were tested for their ability to mediate the hGH-induced immunoprecipitability of JAK2 with phosphotyrosine (APT) antibodies. A rGHR truncated to residue 275 [rGHR-(1-275)], which contains only five cytoplasmic residues, failed to mediate JAK2 APT precipitability in response to hGH. In contrast, WT rGHR; the C-terminal truncations rGHR-(1-542), rGHR-(1-390), and rGHR-(1-317); and the rGHR-(d297-406) deletion mutant maintained this ability. Deletion of the 278-292 box 1-containing region in the context of either rGHR-(d297-406) or WT rGHR eliminated detectable hGH-induced JAK2 APT precipitability. Interestingly, rGHR-(1-292), which includes box 1, was not able to mediate significant hGH-induced JAK2 APT precipitability.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Interaction of the growth hormone receptor cytoplasmic domain with the JAK2 tyrosine kinase. 795 46

Protein-tyrosine kinases (PTKs) of the JAK family have been characterized on the basis of their ability to mediate the rapid induction of transcription of interferon-responsive genes through the stimulation of a class of latent cytoplasmic transcription factors known as signal transducers and activators of transcription (STATs). STAT activation, which has been described as being Ras-independent, requires tyrosine phosphorylation, but STAT transactivating activity is enhanced by phosphorylation on serine as well, probably by extracellular signal-regulated kinase/mitogen-activated protein kinase(s) (ERK/MAPK). STATs can be activated upon binding of ligands to receptor PTKs, to G-protein-linked receptors, and to cytokine receptors. Whether JAKs are required for the activation of signaling pathways other than that leading to STAT activation is not known. The binding of growth hormone (GH) to its receptor (GHR) activates JAK2 and STATs as well as ERK/MAP kinases. We have used a transient transfection system in 293 cells to evaluate the requirement for JAK2 in the activation of ERK2/MAPK by GH. We found that JAK2 is required for GH-simulated activation of ERK2/MAPK. Employing the transient expression of dominant negative forms of H-Ras and Raf-1, we determined that the GHR/JAK2-mediated activation of ERK2/MAPK is dependent on both Ras and Raf. Thus, JAK protein-tyrosine kinases may represent a common component in the activation of the ERK2/MAPK and STAT signaling pathways, which appear to bifurcate upstream of Ras activation but converge with ERK/MAPK phosphorylation of STATs.
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PMID:JAK2, Ras, and Raf are required for activation of extracellular signal-regulated kinase/mitogen-activated protein kinase by growth hormone. 853 33

We are developing a genetic map of the dog based partly upon markers contained within known genes. In order to facilitate the development of these markers, we have used polymerase chain reaction (PCR) primers designed to conserved regions of genes that have been sequenced in at least two species. We have refined the method for designing primers to maximize the number that produce successful amplifications across as many mammalian species as possible. We report the development of primer sets for 11 loci in detail: CFTR, COL10A1, CSFIR, CYP1A1, DCN1, FES, GHR, GLB1, PKLR, PVALB, and RB1. We also report an additional 75 primer sets in the appendices. The PCR products were sequenced to show that the primers amplify the expected canine genes. These primer sets thus define a class of gene-specific sequence-tagged sites (STSs). There are a number of uses for these STSs, including the rapid development of various linkage tools and the rapid testing of genomic and cDNA libraries for the presence of their corresponding genes. Six of the eleven gene targets reported in detail have been proposed to serve as "anchored reference loci" for the development of mammalian genetic maps [O'Brien, S. J., et al., Nat. Genet. 3:103, 1993]. The primer sets should cover a significant portion of the canine genome for the development of a linkage map. In order to determine how useful these primer sets would be for the other genome projects, we tested the 11 primer sets on the DNA from species representing five mammalian orders. Eighty-four percent of the gene-species combinations amplified successfully. We have named these primer sets "universal mammalian sequence-tagged sites" because they should be useful for many mammalian genome projects.
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PMID:Gene-specific universal mammalian sequence-tagged sites: application to the canine genome. 889 53

GH has long been known as a regulator of body growth and metabolism, yet its mechanism of action at the cellular level has been elusive. We have recently shown that GH promotes the rapid association of GH receptor with the tyrosine kinase JAK2, activates JAK2, and promotes the tyrosyl phosphorylation of both JAK2 and GH receptor. This suggests that the initial signalling event in GH action is the activation of JAK2 which in turn phosphorylates tyrosines within JAK2 and GH receptor. We have identified a number of proteins that appear to bind to these phosphotyrosines in GH receptor/ JAK2 complexes. These proteins in turn become phosphorylated on tyrosines, resulting in their activation. These proteins include: 1) the signal transducers and activators of transcriptions (Stats) 1, 3 and 5 which have been implicated as regulators of transcription of a variety of genes; 2) the insulin receptor substrates (IRS) 1 and 2, which are believed to mediate some of the metabolic effects of GH; and 3) Shc proteins which lie upstream of Ras and the mitogen activator kinases (MAP) designated ERKs 1 and 2, proteins implicated in the regulation of cellular growth and/or differentiation. These various proteins work in concert with each other and with other signalling molecules to elicit the diverse effects of GH. Other hormones and growth factors also activate JAK kinases. Specificity in signalling was investigated by determining whether signalling pathways for particular ligands may be selectively inhibited by hormones or growth factors. Glucocorticoids were found to selectively decrease binding and cellular signalling in response to GH. This decrease appeared to be due to a decrease in the number of GH receptors in the plasma membrane. Using truncated and mutated GHR, two regions of the GH receptor were identified required for the inhibitory effect of glucocorticoids. Interestingly, they appeared to differ from the region required for GH-induced internalization. Hence, a large amount of insight into signalling by GH has been obtained during the 3 years since JAK2 was identified as a signalling molecule for GH and other ligands that bind to members of the cytokine receptor family. This new insight, and the insight that will continue to be gained in the next few years should enable the design of new and better therapeutic uses of GH and the other ligands that bind to JAK kinase-linked receptors.
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PMID:Signalling pathway of GH. 907 44

GH is known to activate JAK2 tyrosine kinase and members of the Stat family of transcription factors, including Stats 1, 3, and 5. The recent observation that at least two Stat5 proteins (Stat5A and Stat5B) exist in mouse and human, raises the question of whether GH activates both Stat5A and Stat5B and, if so, whether the requirements for activation are the same. An initial report investigating this issue demonstrated GH-dependent activation of Stat5A but not Stat5B. In this paper, we demonstrate (in COS cells expressing rat GH receptor (rGHR) and either Stat5A or Stat5B, 3T3-F442A fibroblasts, and CHO cells expressing rGHR) that GH induces tyrosyl phosphorylation of both Stat5A and Stat5B. Similar time courses of phosphorylation were observed for the two proteins. Interestingly, the pattern of observed bands differs for the two forms of Stat5. Two closely migrating Stat5A bands can be detected in cells treated with or without GH. Both of these bands become tyrosyl phosphorylated in response to GH. Three species of Stat5B are observed in untreated cells. An additional, more slowly migrating Stat5B band, appears upon treatment with GH. The three more slower migrating Stat5B bands observed in response to GH contain phosphorylated tyrosyl residues. We further demonstrate that GH induces binding of Stat5A and Stat5B, as well as Stat1, to the GAS-like element in the beta-casein promoter. We and others have demonstrated previously that specific regions of GHR are required for GH-dependent activation of what is here identified as Stat5B. To gain insight into the mechanism by which GH promotes tyrosyl phosphorylation of Stat5A, GH-dependent tyrosyl phosphorylation of Stat5A was examined in CHO cells expressing truncated and mutated rGHR. The results indicate that Stat5A and Stat5B require the same regions of rGHR for maximal activation by GH: the C-terminal half of the cytoplasmic domain; tyrosines 333 and/or 338 in the N-terminal half of the cytoplasmic domain; and the regions required for JAK2 activation. To dissect further the mechanism by which GH activates Stat5A and B, the requirement for JAK2 in GH-dependent Stat5 tyrosyl phosphorylation was assessed using JAK2-deficient cells expressing GHR (gamma2A-GHR) and the wild-type parental cell line expressing GHR (2C4-GHR). GH-induced tyrosyl phosphorylation of Stat5B in 2C4-GHR cells but not in the JAK2 deficient, gamma2A-GHR cells, indicating that JAK2 is required for GH-dependent tyrosyl phosphorylation of Stat5B. Western blotting revealed that Stat5A is not expressed in this cell type. Taken together, these findings suggest that: 1) GH activates both Stat5A and Stat5B in several cell types; 2) the pattern of bands observed differs for Stat5A and Stat5B; 3) GH-dependent tyrosyl phosphorylation of Stat5A requires specific regions of GHR, and these requirements are the same as for Stat5B; and 4) JAK2 kinase is required for GH-dependent tyrosyl phosphorylation of Stat5B and, most likely, Stat5A.
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PMID:Growth hormone-induced tyrosyl phosphorylation and deoxyribonucleic acid binding activity of Stat5A and Stat5B. 923 97

Activation of the tyrosine kinase JAK2 is an essential step in cellular signaling by growth hormone (GH) and multiple other hormones and cytokines. Murine JAK2 has a total of 49 tyrosines which, if phosphorylated, could serve as docking sites for Src homology 2 (SH2) or phosphotyrosine binding domain-containing signaling molecules. Using a yeast two-hybrid screen of a rat adipocyte cDNA library, we identified a splicing variant of the SH2 domain-containing protein SH2-B, designated SH2-Bbeta, as a JAK2-interacting protein. The carboxyl terminus of SH2-Bbeta (SH2-Bbetac), which contains the SH2 domain, specifically interacts with kinase-active, tyrosyl-phosphorylated JAK2 but not kinase-inactive, unphosphorylated JAK2 in the yeast two-hybrid system. In COS cells coexpressing SH2-Bbeta or SH2-Bbetac and murine JAK2, both SH2-Bbetac and SH2-Bbeta coimmunoprecipitate to a significantly greater extent with wild-type, tyrosyl-phosphorylated JAK2 than with kinase-inactive, unphosphorylated JAK2. SH2-Bbetac also binds to immunoprecipitated wild-type but not kinase-inactive JAK2 in a far Western blot. In 3T3-F442A cells, GH stimulates the interaction of SH2-Bbeta with tyrosyl-phosphorylated JAK2 both in vitro, as assessed by binding of JAK2 in cell lysates to glutathione S-transferase (GST)-SH2-Bbetac or GST-SH2-Bbeta fusion proteins, and in vivo, as assessed by coimmunoprecipitation of JAK2 with SH2-Bbeta. GH promoted a transient and dose-dependent tyrosyl phosphorylation of SH2-Bbeta in 3T3-F442A cells, further suggesting the involvement of SH2-Bbeta in GH signaling. Consistent with SH2-Bbeta being a substrate of JAK2, SH2-Bbetac is tyrosyl phosphorylated when coexpressed with wild-type but not kinase-inactive JAK2 in both yeast and COS cells. SH2-Bbeta was also tyrosyl phosphorylated in response to gamma interferon, a cytokine that activates JAK2 and JAK1. These data suggest that GH-induced activation and phosphorylation of JAK2 recruits SH2-Bbeta and its associated signaling molecules into a GHR-JAK2 complex, thereby initiating some as yet unidentified signal transduction pathways. These pathways are likely to be shared by other cytokines that activate JAK2.
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PMID:Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling. 934 27

We have previously described two families (H and M) with GH binding protein-positive Laron Syndrome (LS), proposed to have one or more post GHR signaling defects. In the present study, we have examined whether the signal transducers and activators of transcription (STAT) pathway is activated by GH in skin fibroblast cultures established from these LS children, to determine the level(s) at which GH insensitivity has occurred. On immunoblots, both normal and LS fibroblasts express JAK2 and STATs 1, 3, and 5. GH induced rapid tyrosine phosphorylation of a protein at approximately 93 kDa in normal fibroblasts, and Western blotting with STAT-specific antibodies revealed STAT5 activation (phosphorylation) by GH. To determine further the identity and the DNA binding characteristics of the STAT proteins that were activated by GH, EMSAs were performed using three DNA elements known to bind STAT proteins; m67, the high affinity c-sis-inducible element (SIE), the interferon response element (IRE), and the lactogenic hormone-responsive region (LHRR). GH failed to induce protein binding to the SIE or IRE in normal skin fibroblasts but did induce the formation of a specific complex with the LHRR. Induction by GH of this LHRR/protein complex, which could be supershifted partially by anti-STAT1 antisera and completely by anti-STAT5 antisera, was transient, maximal between 10 and 30 min and reduced by 60 min. GH also induced distinct LHRR/protein complexes in mouse 3T3-F442A fibroblasts and in human IM-9 lymphocytes, but supershift analysis revealed that these complexes contained STAT5 but not STAT1. Whereas no binding to the LHRR was observed in GH-treated H fibroblasts, GH induced binding to this element in M fibroblasts. These results demonstrate that 1) the JAK-STAT pathway is activated by GH in normal fibroblasts and that STATs 1 and 5 have a role in GH-dependent signaling in these cells; 2) GH activation of DNA/STAT binding is cell type- and species-specific; and 3) GH failed to activate the STAT pathway in H fibroblasts but induced STAT signaling in M fibroblasts, indicating that the site of GH resistance in the latter is likely to be located within another GH signaling pathway. These fibroblast cultures therefore provide unique models with which to further our understanding of the mechanisms of human GH signaling.
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PMID:Activation of the signal transducers and activators of transcription signaling pathway by growth hormone (GH) in skin fibroblasts from normal and GH binding protein-positive Laron Syndrome children. 942 93

The growth hormone (GH) receptor (GHR) binds GH in its extracellular domain and transduces activating signals via its cytoplasmic domain. Both GH-induced GHR dimerization and JAK2 tyrosine kinase activation are critical in initiation of GH signaling. We previously described a rapid GH-induced disulfide linkage of GHRs in human IM-9 cells. In this study, three GH-induced phenomena (GHR dimerization, GHR disulfide linkage, and enhanced GHR-JAK2 association) were examined biochemically and immunologically. By using the GH antagonist, G120K, and an antibody recognizing a dimerization-sensitive GHR epitope, we demonstrated that GH-induced GHR disulfide linkage reflects GH-induced GHR dimerization. GH, not G120K, promoted both GHR disulfide linkage and enhanced association with JAK2. Measures that diminished GH-dependent JAK2 and GHR tyrosine phosphorylation diminished neither GH-induced GHR disulfide linkage nor GH-enhanced GHR-JAK2 association. By using both transient and stable expression systems, we determined that cysteine 241 (an unpaired extracellular cysteine) was critical for GH-induced GHR disulfide linkage; however, GH-induced GHR dimerization, GHR-JAK2 interaction, and GHR, JAK2, and STAT5 tyrosine phosphorylation still proceeded when this cysteine residue was mutated. We conclude GH-induced GHR disulfide linkage is not required for GHR dimerization, and activation and GH-enhanced GHR-JAK2 association depends more on GHR dimerization than on GHR and/or JAK2 tyrosine phosphorylation.
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PMID:Disulfide linkage of growth hormone (GH) receptors (GHR) reflects GH-induced GHR dimerization. Association of JAK2 with the GHR is enhanced by receptor dimerization. 1055 77

A signaling pathway was delineated by which GH promotes cell survival. Experiments were performed in human leukemic cells (HL-60) and Chinese hamster ovary (CHO) cells. In HL-60 cells, GH treatment reduced starvation-induced cell death. In contrast, when HL-60 cells were treated with an anti-GH antibody, cell survival was sharply reduced. In CHO cells stably expressing either the wild-type (wtGHR) or a truncated form (delta454GHR) of the GH receptor in which GH induces a sustained activation of the receptor-associated tyrosine kinase JAK2, we found that GH stimulation inhibited programmed cell death induced by withdrawal of survival factors. This effect was enhanced in cells expressing the truncated form. In contrast, GH did not affect cell survival in CHO cells transfected with either the empty vector or a mutated GHR unable to transduce the signal (4P/AGHR). We also showed that the inhibitory action of GH on apoptosis is probably mediated via stimulation of the serine-threonine kinase Akt, as 1) GH treatment induces a prompt phosphorylation of Akt; and 2) GH effects on cell survival are abolished by transfection of an Akt mutant that exhibits dominant negative function. Experiments with pharmacological inhibitors demonstrated that GH-induced Akt phosphorylation is dependent on phosphoinositide 3-kinase activation. In contrast, we found no changes in Bcl-2 levels secondary to GHR activation.
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PMID:Activation of growth hormone receptor delivers an antiapoptotic signal: evidence for a role of Akt in this pathway. 1057 61


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