共查询到20条相似文献,搜索用时 46 毫秒
1.
G W Booker A L Breeze A K Downing G Panayotou I Gout M D Waterfield I D Campbell 《Nature》1992,358(6388):684-687
Receptor protein-tyrosine kinases, through phosphorylation of specific tyrosine residues, generate high-affinity binding sites which direct assembly of multienzyme signalling complexes. Many of these signalling proteins, including phospholipase C gamma, GTPase-activating protein and phosphatidylinositol-3-OH kinase, contain src-homology 2 (SH2) domains, which bind with high affinity and specificity to tyrosine-phosphorylated sequences. The critical role played by SH2 domains in signalling has been highlighted by recent studies showing that mutation of specific phosphorylation sites on the platelet-derived growth factor receptor impair its association with phosphatidylinositol-3-OH kinase, preventing growth factor-induced mitogenesis. Here we report the solution structure of an isolated SH2 domain from the 85K regulatory subunit of phosphatidylinositol-3-OH kinase, determined using multidimensional nuclear magnetic resonance spectroscopy. The structure is characterized by a central region of beta-sheet flanked by two alpha-helices, with a highly flexible loop close to functionally important residues previously identified by site-directed mutagenesis. 相似文献
2.
Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis. 总被引:54,自引:0,他引:54
M Mohammadi C A Dionne W Li N Li T Spivak A M Honegger M Jaye J Schlessinger 《Nature》1992,358(6388):681-684
Stimulation of growth factor receptors with tyrosine kinase activity is followed by rapid receptor dimerization, tyrosine autophosphorylation and phosphorylation of signalling molecules such as phospholipase C gamma (PLC gamma) and the ras GTPase-activating protein. PLC gamma and GTPase-activating protein bind to specific tyrosine-phosphorylated regions in growth factor receptors through their src-homologous SH2 domains. Growth factor-induced tyrosine phosphorylation of PLC gamma is essential for stimulation of phosphatidylinositol hydrolysis in vitro and in vivo. We have shown that a short phosphorylated peptide containing tyrosine at position 766 from a conserved region of the fibroblast growth factor (FGF) receptor is a binding site for the SH2 domain of PLC gamma (ref. 8). Here we show that an FGF receptor point mutant in which Tyr 766 is replaced by a phenylalanine residue (Y766F) is unable to associate with and tyrosine-phosphorylate PLC gamma or to stimulate hydrolysis of phosphatidylinositol. Nevertheless, the Y766F FGF receptor mutant can be autophosphorylated, and can phosphorylate several cellular proteins and stimulate DNA synthesis. Our data show that phosphorylation of the conserved Tyr 766 of the FGF receptor is essential for phosphorylation of PLC gamma and for hydrolysis of phosphatidylinositol, but that elimination of this hydrolysis does not affect FGF-induced mitogenesis. 相似文献
3.
Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. 总被引:75,自引:0,他引:75
The critical pathways through which protein-tyrosine kinases induce cellular proliferation and malignant transformation are not well defined. As microinjection of antibodies against p21ras can block the biological effects of both normal and oncogenic tyrosine kinases, it is likely that they require functional p21ras to transmit their mitogenic signals. No biochemical link has been established, however, between tyrosine kinases and p21ras. We have identified a non-catalytic domain of cytoplasmic tyrosine kinases, SH2, that regulates the activity and specificity of the kinase domain. The presence of two adjacent SH2 domains in the p21ras GTPase-activating protein (GAP) indicates that GAP might interact directly with tyrosine kinases. Here we show that GAP, and two co-precipitating proteins of relative molecular masses 62,000 and 190,000 (p62 and p190) are phosphorylated on tyrosine in cells that have been transformed by cytoplasmic and receptor-like tyrosine kinases. The phosphorylation of these polypeptides correlates with transformation in cells expressing inducible forms of the v-src or v-fps encoded tyrosine kinases. Furthermore, GAP, p62 and p190 are also rapidly phosphorylated on tyrosine in fibroblasts stimulated with epidermal growth factor. Our results suggest a mechanism by which tyrosine kinases might modify p21ras function, and implicate GAP and its associated proteins as targets of both oncoproteins and normal growth factor receptors with tyrosine kinase activity. These data support the idea that SH2 sequences direct the interactions of cytoplasmic proteins involved in signal transduction. 相似文献
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The SH2/SH3 adaptor Grb4 transduces B-ephrin reverse signals 总被引:16,自引:0,他引:16
Bidirectional signals mediated by membrane-anchored ephrins and Eph receptor tyrosine kinases have important functions in cell-cell recognition events, including those that occur during axon pathfinding and hindbrain segmentation. The reverse signal that is transduced into B-ephrin-expressing cells is thought to involve tyrosine phosphorylation of the signal's short, conserved carboxy-terminal cytoplasmic domain. The Src-homology-2 (SH2) domain proteins that associate with activated tyrosine-phosphorylated B-subclass ephrins have not been identified, nor has a defined cellular response to reverse signals been described. Here we show that the SH2/SH3 domain adaptor protein Grb4 binds to the cytoplasmic domain of B ephrins in a phosphotyrosine-dependent manner. In response to B-ephrin reverse signalling, cells increase FAK catalytic activity, redistribute paxillin, lose focal adhesions, round up, and disassemble F-actin-containing stress fibres. These cellular responses can be blocked in a dominant-negative fashion by expression of the isolated Grb4 SH2 domain. The Grb4 SH3 domains bind a unique set of other proteins that are implicated in cytoskeletal regulation, including the Cbl-associated protein (CAP/ponsin), the Abl-interacting protein-1 (Abi-1), dynamin, PAK1, hnRNPK and axin. These data provide a biochemical pathway whereby cytoskeletal regulators are recruited to Eph-ephrin bidirectional signalling complexes. 相似文献
6.
Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. 总被引:124,自引:0,他引:124
M Rozakis-Adcock J McGlade G Mbamalu G Pelicci R Daly W Li A Batzer S Thomas J Brugge P G Pelicci 《Nature》1992,360(6405):689-692
The mammalian shc gene encodes two overlapping, widely expressed proteins of 46 and 52K, with a carboxy-terminal SH2 domain that binds activated growth factor receptors, and a more amino-terminal glycine/proline-rich region. These shc gene products (Shc) are transforming when overexpressed in fibroblasts. Shc proteins become phosphorylated on tyrosine in cells stimulated with a variety of growth factors, and in cells transformed by v-src (ref. 2), suggesting that they are tyrosine kinase targets that control a mitogenic signalling pathway. Here we report that tyrosine-phosphorylated Shc proteins form a specific complex with a non-phosphorylated 23K polypeptide encoded by the grb2/sem-5 gene. The grb2/sem-5 gene product itself contains an SH2 domain, which mediates binding to Shc, and is implicated in activation of the Ras guanine nucleotide-binding protein by tyrosine kinases in both Caenorhabditis elegans and mammalian cells. Consistent with a role in signalling through Ras, shc overexpression induced Ras-dependent neurite outgrowth in PC12 cells. These results suggest that Shc tyrosine phosphorylation can couple tyrosine kinases to Grb2/Sem-5, through formation of a Shc-Grb2/Sem-5 complex, and thereby regulate the mammalian Ras signalling pathway. 相似文献
7.
A Tyr/Ser protein phosphatase encoded by vaccinia virus. 总被引:51,自引:0,他引:51
Protein tyrosine phosphorylation is associated with alterations in receptor activity, cellular proliferation and modulation of the cell cycle. Inappropriate tyrosine phosphorylation can lead to unrestrained cell growth and oncogenesis. Enzymes important in tyrosine dephosphorylation have also been described. Protein tyrosine phosphatases (PTPases) consist of two families. There is a receptor-like family of PTPases with an extracellular domain, transmembrane-spanning region and typically two repeated phosphatase domains. Proteins of the non-receptor-like family have a single catalytic phosphatase domain, show a substrate specificity for Tyr phosphate and will not hydrolyse Ser or Thr phosphate. Here we report that the vaccinia virus genome contains an open reading frame which shares amino-acid sequence identity with the PTPases. The purified protein encoded by the vaccinia virus H1 open reading frame expressed in bacteria hydrolyses substrates containing phosphotyrosine and phosphoserine. Mutagenesis of an essential Cys in the vaccinia phosphatase abolishes catalytic activity directed towards both substrates, suggesting that hydrolysis proceeds by a common mechanism. Understanding the function of the H1-encoded protein will help to define the role of the phosphatase in viral replication and pathogenesis. 相似文献
8.
Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. 总被引:55,自引:0,他引:55
The protein-tyrosine kinase activity of the proto-oncogene product p60c-src is negatively regulated by the phosphorylation of a tyrosine residue close to the C terminus, tyrosine 527. The phosphorylation might be catalysed by a so-far-unidentified tyrosine kinase, distinct from p60c-src. Recently we purified a protein-tyrosine kinase that specifically phosphorylates tyrosine 527 of p60c-src from neonatal rat brain. We have now confirmed the specificity of this enzyme by using a mutant p60c-src that has a phenylalanine instead of tyrosine 527, and cloned a complementary DNA that encodes the enzyme. The enzyme is similar to kinases of the src family in that it has two conserved regions, Src-homology regions 2 and 3, upstream of a tyrosine kinase domain. The amino-acid identity of each region is no more than 47%, however, and the enzyme lacks phosphorylation sites corresponding to tyrosines 416 and 527 of p60c-src and has no myristylation signal. These results suggest that this protein-tyrosine kinase, which might negatively regulate p60c-src, represents a new type of tyrosine kinase. 相似文献
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Protein-tyrosine phosphatases (PTPs) are vital for regulating tryosine phosphorylation in many processes, including growth and differentiation. The regulation of receptor-like PTP (RPTP) activity remains poorly understood, but based on the crystal structure of RPTPalpha domain 1 we have proposed that dimerization can negatively regulate activity, through the interaction of an inhibitory 'wedge' on one monomer with the catalytic cleft of domain 1 in the other monomer. Here we show that dimerization inhibits the activity of a full-length RPTP in vivo. We generated stable disulphide-bonded full-length RPTPalpha homodimers by expressing mutants with single cysteines at different positions in the ectodomain juxtamembrane region. Expression of wild-type RPTPalpha and Phe135Cys and Thr141Cys mutants in RPTPalpha-null mouse embryo cells increased dephosphorylation and activity of Tyr 529 in the protein tyrosine kinase c-Src; in contrast, expression of a Pro137Cys mutant did not. Mutation of Pro 210/211 to leucine in the inhibitory wedge of the Pro137Cys mutant restored its ability to activate c-Src, indicating that dimerization may inhibit full-length RPTPalpha activity in a manner stereochemically consistent with RPTPalpha crystal structures. Our results suggest that RPTPalpha activity can in principle be negatively regulated by dimerization in vivo. 相似文献
11.
The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration. 相似文献
12.
Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane 总被引:5,自引:0,他引:5
The receptor for epidermal growth factor (EGF) is a 170,000-180,000 molecular weight single-chain glycoprotein of 1,186 amino acids. Its sequence suggests that it has an external EGF-binding domain, formed by the NH2-terminal 621 amino acids, linked to a cytoplasmic region by a single membrane-spanning segment. In the cytoplasmic portion, starting 50 residues from the membrane, there is a 250-residue stretch similar to the catalytic domain of the src gene family of retroviral tyrosine protein kinases, and, indeed, a tyrosine-specific protein kinase activity intrinsic to the receptor is stimulated when EGF is bound. Increased tyrosine phosphorylation of cellular proteins, detected in A431 cells following EGF binding, may be important in the mitogenic signal pathway. Tumour promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), counteract this increase, as well as causing loss of a high affinity class of EGF binding sites. The major receptor for TPA has been identified as the serine/threonine-specific Ca2+/phospholipid-dependent diacylglycerol-activated protein kinase, protein kinase C. By substituting for diacylglycerol, TPA stimulates protein kinase C. Protein kinase C phosphorylates purified EGF receptor at specific sites, and this reduces EGF-stimulated tyrosine protein kinase activity. TPA treatment of A431 cells increases serine and threonine phosphorylation of the EGF receptor at the same sites, which suggests that the reduction of EGF receptor kinase activity in TPA-treated cells is a consequence of the receptor's phosphorylation by the kinase. We have attempted to identify these phosphorylation sites and show here that protein kinase C phosphorylates threonine 654 in the human EGF receptor. This threonine is in a very basic sequence nine residues from the cytoplasmic face of the plasma membrane in the region before the protein kinase domain; it is thus in a position to modulate signalling between this internal domain and the external EGF-binding domain. 相似文献
13.
SHIWuliang JIAWensuo LIUXin ZHANGShuqiu 《科学通报(英文版)》2004,49(17):1841-1846
Although protein tyrosine phosphatases (PTPases) play an important role in signal transduction in animal cells, little is known about the function of PTPases in higher plants. Hydrogen peroxide (H2O2) and mitogen-activated protein kinases (MAPKs) are the critical components of ABA signaling pathway in guard cells. PTPase is an important regulator of MAPK, which is believed to mediate ABA-induced H2O2 generation in guard cells of Vicia faba L. Here, we investigate the possible role of PTPases in stomatal movement process. Phenylarsine oxide (PAO), a specific inhibitor of PTPases, could prevent ABA or H2O2-induced stomatal closure of Vicia faba L; furthermore, it could promote opening of the stomata closed by ABA or H2O2. The activity of PTPases can be effectively inhibited by PAO and H2O2. DTT had no effect on the PAO-induced inhibition of PTPases activity, but it could relieve the inhibition of H2O2 on PTPases activity. PAO could also inhibit the ABA-induced H2O2 generation in guard cells of V‘wia faba L. These results suggested that PTPases is a critical signaling component in ABA-induced stomatal closure, and serve as targets for H2O2 lying on the signaling pathways downstream of ABA induced H2O2 generation. 相似文献
14.
Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins. 总被引:43,自引:0,他引:43
Many hormones, neurotransmitters and growth factors, on binding to G protein-coupled receptors or receptors possessing tyrosine kinase activity, increase intracellular levels of the second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. This is due to activation of phosphoinositide-specific phospholipase(s) C (PLC), the isozymes of which are classified into groups, alpha, beta, gamma and delta. The beta, gamma and delta groups themselves contain PLC isozymes which have both common and unique structural domains. Only the gamma 1 isozyme has been implicated in a signal transduction mechanism. This involves association with, and tyrosine phosphorylation by, the ligand-bound epidermal growth factor and platelet-derived growth factor receptors, probably by means of the PLC-gamma 1-specific src homology (SH2) domain. Because EGF receptor-mediated tyrosine phosphorylation of PLC-gamma 1 stimulates catalytic activity in vitro and G proteins have been implicated in the activation of PLC, we investigated which PLC isozymes are subject to G protein regulation. We have purified an activated G protein alpha subunit that stimulates partially purified phospholipase C and now report that this G protein specifically activates the beta 1 isozyme, but not the gamma 1 and delta 1 isozymes of phospholipase C. We also show that this protein is related to the Gq class of G protein alpha subunits. 相似文献
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Choi MH Lee IK Kim GW Kim BU Han YH Yu DY Park HS Kim KY Lee JS Choi C Bae YS Lee BI Rhee SG Kang SW 《Nature》2005,435(7040):347-353
Platelet-derived growth factor (PDGF) is a potent mitogenic and migratory factor that regulates the tyrosine phosphorylation of a variety of signalling proteins via intracellular production of H2O2 (refs 1, 2-3). Mammalian 2-Cys peroxiredoxin type II (Prx II; gene symbol Prdx2) is a cellular peroxidase that eliminates endogenous H2O2 produced in response to growth factors such as PDGF and epidermal growth factor; however, its involvement in growth factor signalling is largely unknown. Here we show that Prx II is a negative regulator of PDGF signalling. Prx II deficiency results in increased production of H2O2, enhanced activation of PDGF receptor (PDGFR) and phospholipase Cgamma1, and subsequently increased cell proliferation and migration in response to PDGF. These responses are suppressed by expression of wild-type Prx II, but not an inactive mutant. Notably, Prx II is recruited to PDGFR upon PDGF stimulation, and suppresses protein tyrosine phosphatase inactivation. Prx II also leads to the suppression of PDGFR activation in primary culture and a murine restenosis model, including PDGF-dependent neointimal thickening of vascular smooth muscle cells. These results demonstrate a localized role for endogenous H2O2 in PDGF signalling, and indicate a biological function of Prx II in cardiovascular disease. 相似文献
17.
PDGF induction of tyrosine phosphorylation of GTPase activating protein 总被引:107,自引:0,他引:107
C J Molloy D P Bottaro T P Fleming M S Marshall J B Gibbs S A Aaronson 《Nature》1989,342(6250):711-714
The cascade of biochemical events triggered by growth factors and their receptors is central to understanding normal cell-growth regulation and its subversion in cancer. Ras proteins (p21ras) have been implicated in signal transduction pathways used by several growth factors, including platelet-derived growth factor (PDGF). These guanine nucleotide-binding Ras proteins specifically interact with a cellular GTPase-activating protein (GAP). Here we report that in intact quiescent fibroblasts, both AA and BB homodimers of PDGF rapidly induce tyrosine phosphorylation of GAP under conditions in which insulin and basic fibroblast growth factor (bFGF) are ineffective. Although GAP is located predominantly in the cytosol, most tyrosine-phosphorylated GAP is associated with the cell membrane, the site of p21ras biological activity. These results provide a direct biochemical link between activated PDGF-receptor tyrosine kinases and the p21ras-GAP mitogenic signalling system. 相似文献
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High tyrosine kinase activity in normal nonproliferating cells 总被引:1,自引:0,他引:1
Protein phosphorylation at serine and threonine residues has been implicated in the regulation of many cellular processes. More recently, tyrosine residue phosphorylation has been shown to be associated with stimulation of cell proliferation, including viral transformation and stimulation by epidermal growth factors (EGF), platelet-derived growth factor (PDGF) and other compounds related to cellular growth such as insulin and dimethyl sulphoxide. To compare protein kinases and phosphoproteins of normal and leukaemic human haematopoietic cells in vivo and in vitro, we first have investigated the percentages of phosphoserine, phosphothreonine and phosphotyrosine obtained after hydrolysis of proteins from different blood cell fractions phosphorylated in vitro. We report here that phosphotyrosine formed less than 1% of the soluble fractions from polymorphonuclear cells, mononuclear cells (80% circulating lymphocytes, 20% monocytes), blood platelets and red blood cells (not shown). Surprisingly, high percentages of phosphorylated tyrosine were found only in the particulate fractions from non-proliferating anuclear cells, platelets and red blood cells. 相似文献
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Phospholipase C gamma 1 is a physiological guanine nucleotide exchange factor for the nuclear GTPase PIKE 总被引:4,自引:0,他引:4
Ye K Aghdasi B Luo HR Moriarity JL Wu FY Hong JJ Hurt KJ Bae SS Suh PG Snyder SH 《Nature》2002,415(6871):541-544
Phospholipase C gamma 1 (PLC-gamma 1) hydrolyses phosphatidylinositol-4,5-bisphosphate to the second messengers inositol-1,4,5-trisphosphate and diacylglycerol. PLC-gamma 1 also has mitogenic activity upon growth-factor-dependent tyrosine phosphorylation; however, this activity is not dependent on the phospholipase activity of PLC-gamma 1, but requires an SH3 domain. Here, we demonstrate that PLC-gamma 1 acts as a guanine nucleotide exchange factor (GEF) for PIKE (phosphatidylinositol-3-OH kinase (PI(3)K) enhancer). PIKE is a nuclear GTPase that activates nuclear PI(3)K activity, and mediates the physiological activation by nerve growth factor (NGF) of nuclear PI(3)K activity. This enzymatic activity accounts for the mitogenic properties of PLC-gamma 1. 相似文献