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)

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

Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells expressing wild-type GHR (GHR1-638) or GHR truncated at amino acid 454 (GHR1-454) or 380 (GHR1-380). JAK2 did not associate with GHR in cells expressing GHR truncated at amino acid 294 (GHR1-294) or when amino acids 297-311 containing a proline-rich motif were deleted (GHR delta P) or prolines 300, 301, 303, and 305 in the proline-rich motif were mutated to alanines (GHR4P-->A). Cross-linking 125I-human GH to GHR demonstrated that GHR mutants migrated with the appropriate molecular weight, with the exception of GHR4P-->A which migrated as a protein similar in size to GHR1-294. In studies performed in CHO and RIN-5AH cells, the ability of JAK2 to associate with the mutated GHR was found to correlate with GH-dependent activation of JAK2, tyrosyl phosphorylation of GHR (in the case of GHR1-638 and GHR1-454), and the ability of the GHR to copurify with tyrosine kinase activity. In CHO cells expressing mutated GHR, GH-dependent tyrosyl phosphorylation of cellular proteins (p121, p97, p42, and p39) was dependent on the ability to activate JAK2. No proteins showed increased tyrosyl phosphorylation in CHO cells expressing GHR1-294, GHR4P-->A, or GHR delta P. Deletion of the C-terminal half (amino acids 455-638) of the GHR ablated GH-dependent tyrosyl phosphorylation of p97. Taken together, these results provide strong evidence that the N-terminal quarter of the cytoplasmic domain of GHR and within this region, the proline-rich motif, is required for association of JAK2 with GHR and GH-dependent activation of JAK2, and that tyrosines in the N-terminal half of the cytoplasmic domain of the GHR are phosphorylated by JAK2. The finding that a specific interaction with the C-terminal half of GHR appears to be necessary for p97 phosphorylation indicates that while JAK2 activation may be necessary for a full biological response to GH, it appears not to be sufficient.
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PMID:Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase. 806 15

Protein kinase play important roles in the growth and differentiation of cells. We have isolated cDNA clones from the human megakaryocytic cell line CMK11-5 that encode a novel protein kinase, which we call SPRK (src-homology 3 (SH3) domain-containing proline-rich kinase). The gene sequence predicts an 847-amino acid protein kinase with a unique domain arrangement. An amino-terminal glycine-rich region is followed by an SH3 domain and a kinase domain that is similar to both tyrosine and serine/threonine kinases. Adjacent to the kinase domain are two closely spaced leucine/isoleucine zipper motifs and a stretch of basic amino acids that resembles karyophilic nuclear localization signals. The COOH-terminal half of SPRK is basic, and proline accounts for 24% of the COOH-terminal 216 amino acids. The sprk gene is widely expressed as a 4-kilobase transcript in adult and fetal human tissues. Transfection of 293 cells with a vector encoding an epitope-tagged SPRK results in the expression of a 95-kDa protein. The epitope-tagged SPRK becomes phosphorylated on serine and threonine residues in an in vitro kinase assay, whereas SPRK variants with point mutations in the predicted ATP-binding site fail to become phosphorylated. These data indicate that SPRK has serine/threonine kinase activity. The SH3 domain of SPRK is interrupted by a unique 5-amino acid insert whose location in the SH3 consensus sequence is the same as that of the inserts found in the SH3 domains of neuronal SRC and of the p85 subunit of phosphatidylinositol 3-kinase.
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PMID:Identification and characterization of SPRK, a novel src-homology 3 domain-containing proline-rich kinase with serine/threonine kinase activity. 819 46

The growth hormone receptor (GHR) belongs to the superfamily of transmembrane proteins that includes the prolactin receptor and a number of cytokine receptors. Two forms exist for the GHR: the full-length membrane-bound human receptor is a protein of 620 amino acids with a single transmembrane region; and the GH binding protein (GHBP) is a short soluble from corresponding to the extracellular domain of the full-length receptor. In rodents, GHBP is encoded by a specific mRNA of 1.2-1.5 kb, whereas in man and other species GHBP is believed to result from proteolytic cleavage of the membrane receptor. Growth hormone binding protein prolongs the half-life of GH but other functions for GHBP remain to be demonstrated. Recombinant GHBP complexed to human GH shows a 2:1 stoichiometric crystal structure. Growth hormone-induced dimerization of the cell surface GHR appears to be a prerequisite for biological activity of the hormone. JAK2 has been identified as a tyrosine kinase associated with GHR and other receptors of the superfamily. Binding of GH to its receptor results in dimerization of the GHR, phosphorylation of JAK2 and of the GHR. Other substrates for JAK2 have to be identified. Transcription factors belonging to the STAT (signal transducers and activators of transcriptions) family are involved in the transcriptional effects of GH. The activity of mutants of the GHR has been measured in functional tests to identify sequences of the cytoplasmic domain of the receptor that are important for signal transduction. A proline-rich sequence, called Box I, conserved among members of the receptor family has been shown to be crucial for GH effects on gene transcription. MAP kinase activity and cell proliferation. The C-terminal region of the GHR is required for tyrosine phosphorylation of the receptor and for a hormonal effect on gene transcription, whereas only 46 membrane proximal amino acids of the cytoplasmic domain are necessary for activation of JAK2 and transduction of the GH proliferative signal. Much work remains to be done to identify other protein kinases and signalling molecules involved in the mechanism of action of GH.
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PMID:Growth hormone receptor: structure and signal transduction. 854 48

Mutational analysis of the proximal transmembrane region of the cytoplasmic domain of the GH receptor (GHR) allowed us to characterize box 1, a proline-rich sequence of eight amino acids, which has been shown to be critical for signal transduction of many cytokine receptors. Mutants of the box 1 region of the rat GHR were studied for their ability to initiate the phosphorylation of JAK2 and the proliferation of stably transfected BAF B03 cells and also the activation of Spi 2.1 gene transcription in transiently transfected Chinese hamster ovary (CHO) cells. Convergence of effects of the box 1 mutants on JAK 2 phosphorylation, cell proliferation, and gene transcription was found. Our results suggest that no single amino acid in the box 1 sequence is essential for signaling and that the last two prolines (PXP motif) and the hydrophobic residues are necessary for integrity of box 1. Box 1 represents a structural determinant, potentially able to provide an interaction between JAK2 and the receptor; this interaction could be direct or indirect via an adaptor protein.
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PMID:The proline-rich region of the GH receptor is essential for JAK2 phosphorylation, activation of cell proliferation, and gene transcription. 861 6

Binding of alpha interferon (IFNalpha) to its receptors induces rapid tyrosine phosphorylation of the receptor subunits IFNaR1 and IFNaR2, the TYK2 and JAK1 tyrosine kinases, and the Stat1 and Stat2 transcription factors. Previous studies have demonstrated that TYK2 directly and specifically binds to and tyrosine phosphorylates IFNaR1 in vitro. We now report a detailed analysis of the TYK2 binding domain on the IFNaR1 subunit. First, we used an in vitro binding assay to identify the TYK2 binding motif in IFNaR1 as well as the critical residues within this region. The most striking feature is the importance of a number of hydrophobic and acidic residues. A minor role is also ascribed to a region resembling the proline-rich "box 1" sequence. In addition, mutations which disrupt in vitro binding also disrupt the coimmunoprecipitation of the receptor and TYK2. We also provide direct evidence that the binding region is both necessary and sufficient to activate TYK2 in vivo. Specifically, mutations in the binding domain act in a dominant-negative fashion to inhibit the IFNalpha-induced tyrosine phosphorylation of TYK2 and Stat2. Further, introduction of dimerized glutathione S-transferase-IFNaR1 fusion proteins into permeabilized cells is sufficient to induce phosphorylation of TYK2 and the receptor, confirming the role of the binding domain in IFNalpha signal transduction. These studies provide clues to the sequences determining the specificity of the association between JAK family tyrosine kinases and cytokine receptors as well as the functional role of these kinases in cytokine signal transduction.
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PMID:Molecular characterization of an alpha interferon receptor 1 subunit (IFNaR1) domain required for TYK2 binding and signal transduction. 862 73

p130(Cas) (crk associated substrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3 domain, and a proline-rich domain. The structure of p130(Cas) suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in cells is unknown. In this report we show that p130(Cas) is localized to focal adhesions. We demonstrate that p130(Cas) associates both in vitro and in vivo with pp125(FAK) (focal adhesion kinase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130(Cas) also associates with pp41/43(FRNK) (pp125(FAK)-related, non-kinase), an autonomously expressed form of pp125(FAK) composed of only the C-terminal noncatalytic domain. We show that the association of p130(Cas) with pp125(Fak) and pp41/43(FRNK) is direct, and is mediated by the binding of the SH3 domain of p130(Cas) to a proline-rich sequence present in both the C terminus of pp125(FAK) and in pp41/43(FRNK). In agreement with recent studies we show that p130(Cas) is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130(Cas) with pp125(FAK), a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.
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PMID:p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase. 866 21

Budding in Saccharomyces cerevisiae follows a genetically programmed pattern of cell division which can be regulated by external signals. On the basis of the known functional conservation between a number of mammalian oncogenes and antioncogenes with genes in the yeast budding pathway, we used enhancement of pseudohyphal budding in S. cerevisiae by human proteins expressed from a HeLa cDNA library as a morphological screen to identify candidate genes that coordinate cellular signaling and morphology. In this report, we describe the isolation and characterization of human enhancer of filamentation 1 (HEF1), an SH3-domain-containing protein that is similar in structure to pl30cas, a recently identified docking protein that is a substrate for phosphorylation by a number of oncogenic tyrosine kinases. In contrast to p130cas, the expression of HEF1 appears to be tissue specific. Further, whereas p130cas is localized predominantly at focal adhesions, immunofluorescence indicates that HEF1 localizes to both the cell periphery and the cell nucleus and is differently localized in fibroblasts and epithelial cells, suggesting a more complex role in cell signalling. Through immunoprecipitation and two-hybrid analysis, we demonstrate a direct physical interaction between HEF1 and p130cas, as well as an interaction of the SH3 domain of HEF1 with two discrete proline-rich regions of focal adhesion kinase. Finally, we demonstrate that as with p130cas, transformation with the oncogene v-abl results in an increase in tyrosine phosphorylation on HEF1, mediated by a direct association between HEF1 and v-Abl. We anticipate that HEF1 may prove to be an important linking element between extracellular signalling and regulation of the cytoskeleton.
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PMID:Human enhancer of filamentation 1, a novel p130cas-like docking protein, associates with focal adhesion kinase and induces pseudohyphal growth in Saccharomyces cerevisiae. 866 48

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.
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PMID:Critical proline residues of the cytoplasmic domain of the IL-5 receptor alpha chain and its function in IL-5-mediated activation of JAK kinase and STAT5. 867 9

The tyrosine kinase Itk/Tsk is a T cell specific analog of Btk, the tyrosine kinase defective in the human immunodeficiency X-linked agammaglobulinemia and in xid mice. T lymphocytes from Itk-deficient mice are refractory to mitogenic stimuli delivered through the T cell receptor (TCR). To gain insights into the biochemical role of Itk, the binding properties of the Itk SH3 domain were examined. An optimal Itk SH3 binding motif was derived by screening biased phage display libraries; peptides based on this motif bound with high affinity and selectivity to the Itk SH3 domain. Initial studies with T cell lysates indicated that the Itk SH3 domain bound Cbl, Fyn, and other tyrosine phosphoproteins from TCR-stimulated Jurkat cells. Under conditions of increased detergent stringency Sam 68, Wiskott-Aldrich Syndrome protein, and hnRNP-K, but not Cbl and Fyn, were bound to the Itk SH3 domain. By examining the ability of different SH3 domains to interact with deletion variants of Sam 68 and WASP, we demonstrated that the Itk-SH3 domain and the SH3 domains of Src family kinases bind to overlapping but distinct sets of proline-rich regions in Sam 68 and WASP.
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PMID:Identification of Itk/Tsk Src homology 3 domain ligands. 881 Mar 41


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