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1.
Cell Signal ; 13(7): 475-81, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11516622

RESUMO

Vav2 is a member of the Vav family that serves as guanine nucleotide exchange factors (GEFs) for the Rho family of Ras-related GTPases. Unlike Vav1, whose expression is restricted to cells of hematopoietic origin, Vav2 is broadly expressed. Recently, Vav2 has been identified as a substrate for the EGF receptor. Here, we show that in EGF-treated COS7 cells Vav2 is phosphorylated on tyrosine residues and associates with the EGF receptor. In addition, introducing point mutations into the SH2 domain of green fluorescens protein (GFP)-Vav2 fusion protein leads to the loss of Vav2 tyrosine phosphorylation in response to EGF. To investigate further the mechanism of Vav2 phosphorylation, N-terminal (NT) domain of Vav2 was transiently expressed in COS7 cells as GFP fusion protein. Whereas the NT domain of Vav2 is a preferred substrate for the activated EGF receptor in vitro, we could not detect tyrosine phosphorylation of the GFP-NT construct in EGF-treated cells. However, when the SH2 domain of Vav2 was fused to its NT domain, NT domain proved to be a substrate for the EGF receptor in vivo. These data suggest that membrane-targeting of Vav2 through its SH2 domain is an important event in the phosphorylation and activation of Vav2 in response to EGF.


Assuntos
Receptores ErbB/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Mutação , Proteínas Oncogênicas/genética , Fosforilação , Fosfotirosina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais
2.
Biochim Biophys Acta ; 1422(2): 187-204, 1999 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-10393272

RESUMO

SH2/SH3 domain-containing adaptor proteins play a critical role in regulating tyrosine kinase signalling pathways. The major function of these adaptors, such as Grb2, Nck, and Crk, is to recruit proline-rich effector molecules to tyrosine-phosphorylated kinases or their substrates. In recent years dozens of novel proteins have emerged that are capable of associating with the SH2 and the SH3 domains of adaptors. In this review, the author attempts to summarise these novel binding partners of Grb2, Nck, and Crk, and to discuss current controversies regarding function and regulation of protein multicomplexes held together by SH2/SH3 adaptor molecules at the plasma membrane.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Domínios de Homologia de src , Subunidades alfa do Complexo de Proteínas Adaptadoras , Proteínas Adaptadoras de Transporte Vesicular , Animais , Proteína Adaptadora GRB2 , Humanos , Proteínas de Membrana/química , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Conformação Proteica , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src
3.
Cell Signal ; 11(4): 253-62, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10372803

RESUMO

The Nck adaptor protein comprises a single C-terminal SH2 domain and three SH3 domains. The domain structure of Nck suggests that Nck links tyrosine kinase substrates to proteins containing proline-rich motifs. Here we show that Bcr/Abl tyrosine kinase, and three tyrosine phosphorylated proteins (115, 120 and 155 kDa) are co-immunoprecipitated with antibody against Nck from lysates of the human leukaemia cell line K562. By means of affinity purification with the Nck-binding phosphopeptide EPGPY(P)AQPSV, we could also detect the association of endogenous Nck with the proto-oncogene product Cbl. An investigation of the nature of interactions revealed that Bcr/Abl, Cbl, and the 155-kDa tyrosine phosphotyrosine bind exclusively to the SH3 domains of Nck. In addition, none of the single SH3 domains of Nck expressed as glutathione-S-transferase (GST) fusion proteins is able to interact with the proline-rich ligands. However, combined first and second SH3 domains have the capacity to bind Bcr/Abl, Chl and p155. Mutations of conserved tryptophan to Lysine in either of the combined first and second SH3 domains completely abolish ligand binding. These data suggest that cooperation exists among the SH3 domains of Nck for a high-affinity binding of proteins containing proline-rich motifs.


Assuntos
Proteínas Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal , Sítios de Ligação , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Células K562 , Ligantes , Lisina/genética , Lisina/metabolismo , Mutagênese Sítio-Dirigida , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Proteínas Recombinantes de Fusão/metabolismo , Triptofano/genética , Triptofano/metabolismo
4.
Eur J Immunol ; 29(4): 1068-75, 1999 04.
Artigo em Inglês | MEDLINE | ID: mdl-10229072

RESUMO

The Nck adaptor protein links tyrosine kinases or their substrates to proteins containing proline-rich motifs. Here we show that in activated T cells two tyrosine phosphoproteins of 75 and 120 kDa are co-immunoprecipitated with polyclonal antibodies against Nck. Analysis of Nck immunoprecipitates with various candidate antibodies revealed that the 75-kDa tyrosine phosphoprotein is the SH2 domain-containing leukocyte protein referred to as SLP-76. In vitro experiments show that the interaction between Nck and SLP-76 is mediated via the Nck SH2 domain. Using specific phosphopeptides corresponding to the major tyrosine phosphorylation sites of SLP-76, it was found that Y113 and Y128 phosphopeptides could compete binding of SLP-76 to the SH2 domain of Nck. In addition, the 120-kDa tyrosine phosphoprotein was recognized by an antibody raised against Cbl, a proto-oncogene product that has been previously found to be associated with Nck. These results suggest that the Nck adaptor protein interacts with key signaling molecules and may play an important role in activation of T lymphocytes.


Assuntos
Proteínas Oncogênicas/análise , Fosfoproteínas/análise , Linfócitos T/química , Tirosina/metabolismo , Ubiquitina-Proteína Ligases , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Humanos , Células Jurkat , Ativação Linfocitária , Dados de Sequência Molecular , Proteínas Oncogênicas/metabolismo , Fosfoproteínas/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Receptores de Antígenos de Linfócitos T/fisiologia , Domínios de Homologia de src
5.
Cell Signal ; 11(1): 25-9, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10206341

RESUMO

One of the adaptor proteins, Nck, comprises a single SH2 domain and three SH3 domains that are important in protein-protein interactions. The in vivo association of Nck with the guanine nucleotide exchange factor Sos has been well documented; however, the precise nature of the interaction is unclear. To determine which SH3 domains are involved in the Nck-Sos interaction, individual SH3 domains of Nck were generated as glutathione S-transferase fusion proteins. We found that exclusively the third (C-terminal) SH3 domain of Nck has the ability to bind to Sos. In addition, in [35S]methionine labelled K562 cells, a 100,000 Mr protein was found to be associated with the third SH3 domain of Nck. This protein was identified as dynamin, a GTP-binding protein that has been implicated in clathrin-coated vesicle formation. Dynamin and Nck co-precipitated when cell lysates were immunoprecipitated with anti-Nck antibody. These data suggest that Nck may contribute to Ras activation and the function of dynamin in membrane trafficking through its third SH3 domain.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas/metabolismo , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Dinaminas , Fatores de Troca do Nucleotídeo Guanina , Humanos , Células K562 , Dados de Sequência Molecular , Proteínas Oncogênicas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores ras de Troca de Nucleotídeo Guanina
6.
Cell Signal ; 11(12): 885-90, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10659996

RESUMO

MAP kinase cascade-dependent responses were investigated during scattering of HepG2 human hepatoma cells stimulated by HGF or phorbol ester. Inhibition of phosphatidylinositol 3-kinase with LY294002 prevented completely the dissociation of cells. Inhibition of MAP kinase kinase (MEK) with PD98059 prevented the development of characteristic morphological changes associated with cell migration. EGF, which failed to induce cell scattering, caused a short-term increase in the phosphorylation of Erk1/Erk2 MAP kinases. On the contrary, HGF or phorbol ester stimulated the phosphorylation of MAP kinases for a long time. Experiments performed with LY294002 indicated that phosphatidylinositol 3-kinase contributed to the HGF-stimulated phosphorylation of Erk1/Erk2. This finding was confirmed by the demonstration that the MAP kinase cascade-dependent expression of a high-Mr (>300 kDa) protein pair appearing in the course of cell scattering was inhibited by LY294002 in HGF-induced cells but was not inhibited in phorbol ester-treated cells.


Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Ubiquitina-Proteína Ligases , Carcinoma Hepatocelular/patologia , Movimento Celular/efeitos dos fármacos , Cromonas/farmacologia , Cicloeximida/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Morfolinas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-cbl , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos
7.
Biochem Pharmacol ; 54(7): 781-9, 1997 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-9353132

RESUMO

The effects of two different tyrosine kinase inhibitors (genistein and erbstatin analog) and an inhibitor (2'-amino-3'-methoxyflavone; PD98059) of the mitogen-activated protein (MAP) kinase kinase on the primary granule exocytosis and superoxide (O2.-) production of human neutrophil granulocytes were compared. The effector responses induced by stimulation of the chemotactic receptors by formyl-methionyl-leucyl-phenylalanine and platelet-activating factor were blocked both by genistein and erbstatin analog. In contrast, degranulation and O2.- production triggered by the activation of protein kinase C with phorbol-12-myristate-13-acetate were reduced by erbstatin analog but not by genistein. This inhibitory pattern was observed in both effector responses, but the sensitivity of O2.- production toward tyrosine kinase inhibition was markedly higher than that of degranulation. PD98059 caused no considerable effect on any of the above responses. The data presented indicate that tyrosine kinases are involved not only in the respiratory burst but also in the organization of the degranulation response of neutrophil granulocytes. It is suggested that several tyrosine kinases of different inhibitor sensitivity may participate in the transduction of extracellular signals. However, activation of the MAP kinase cascade does not appear to be involved in either of the investigated biological responses of the neutrophils.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Degranulação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neutrófilos/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Superóxidos/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Flavonoides/farmacologia , Genisteína/farmacologia , Humanos , Hidroquinonas/farmacologia , Neutrófilos/fisiologia , Fosforilação , Explosão Respiratória/efeitos dos fármacos
8.
J Biol Chem ; 272(26): 16670-8, 1997 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-9195983

RESUMO

To investigate the signal transduction of osmotic stress, we examined hypertonicity-induced tyrosine phosphorylations in Chinese hamster ovary cells. Hyperosmosis elicited characteristic phosphotyrosine accumulation in at least 3 proteins (approximately 42, approximately 85, and approximately 120 kDa). The most prominent response occurred in the 85-kDa band (p85) whose phosphorylation was rapid, sustained, apparent already at mild hypertonicity (350 mosM), proportional to the extracellular osmotic concentration, and reversible. Hyperosmotic environment could not induce tyrosine phosphorylation if cell shrinkage was prevented by nystatin and appropriately composed media. Conversely, isotonic shrinkage caused strong tyrosine phosphorylation. Thus, the initial signal is a decrease in cell volume and not an increase in the intra- or extracellular osmotic concentration, or a rise in cytosolic K+ and Cl- levels. Tyrosine phosphorylation of p85 was not due to the hypertonicity-induced protein kinase C-dependent stimulation of the extracellular signal-regulated protein kinase, nor to the activation of stress-activated protein kinases. Tonicity-responsive proteins interacted with Grb2-glutathione S-transferase fusion proteins: the 120-kDa protein complexed with the SH2 and both SH3 domains, whereas p85 associated with the SH2 and the N-terminal SH3 domains of the adapter. Tyrosine phosphorylation of p85 is a sensitive indicator of reduced intracellular hydration and might signify a hitherto unrecognized, early volume-dependent signaling event.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Tirosina/metabolismo , Animais , Células CHO , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cricetinae , Proteína Adaptadora GRB2 , Proteína Quinase 1 Ativada por Mitógeno , Pressão Osmótica , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Domínios de Homologia de src
9.
J Biol Chem ; 271(11): 6159-63, 1996 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-8626404

RESUMO

Several recent studies have demonstrated that Grb2, composed entirely of SH2 and SH3 domains, serves as an adaptor protein in tyrosine kinase signaling pathways. Cb1, the protein product of c-cbl proto-oncogene, has been reported to be phosphorylated on tyrosine residues upon T cell receptor (TCR) engagement. Here we show that in unstimulated Jurkat cells Cbl is co-immunoprecipitated with monoclonal antibody against Grb2. However, in lymphocytes activated through the TCR, Cbl loses its ability to bind to Grb2 precipitated either with anti-Grb2 antibody or with an immobilized tyrosine phosphopeptide, Y1068-P, derived from the epidermal growth factor receptor. In vitro studies confirm that the ability of Cb1 to bind to both SH3 domains of Grb2 is strongly reduced in activated T lymphocytes. Investigation of the time course of Cbl dissociation from Grb2 reveals that it is transient and correlates with the kinetics of tyrosine phosphorylation of Cbl. Moreover, Cb1 is co-immunoprecipitated with Crk, another SH2/SH3 domain-containing protein, upon TCR stimulation. Tyrosine-phosphorylated Cbl binds exclusively to the SH2 domain of Crk. These results suggest that different adaptor proteins may have different roles in the regulation of c-cbl proto-oncogene product.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Proteína Adaptadora GRB2 , Humanos , Cinética , Ativação Linfocitária , Dados de Sequência Molecular , Fosforilação , Proteínas/química , Proteínas/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-cbl , Proteínas Proto-Oncogênicas c-crk , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tirosina/metabolismo , Domínios de Homologia de src
10.
Oncogene ; 11(7): 1327-31, 1995 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-7478553

RESUMO

Formation of a complex of the nucleotide exchange factor Sos, the SH2 and SH3 containing adaptor protein Grb2/Sem-5 and tyrosine phosphorylated EGF receptor and Shc has been implicated in the activation of Ras by epidermal growth factor (EGF) in fibroblasts: related mechanisms for activation of Ras operate in other cell types. An increase in the apparent molecular weight of Sos has been reported to occur after several minutes of receptor stimulation due to phosphorylation by mitogen-activated protein (MAP) kinases. We report here that treatment of human peripheral blood T lymphoblasts with phorbol esters causes a similar shift in mobility of Sos. This modification of Sos does not alter its ability to bind Grb2, but correlates with strong inhibition of the binding of the Sos/Grb2 complex to tyrosine phosphorylated sequences, either a tyrosine phosphopeptide in cell lysates or p36 in intact cells. This effect, along with the mobility shift of Sos, can be mimicked in vitro by phosphorylation of Sos by the mitogen-activated protein kinase, ERK1. A novel negative feedback mechanism therefore exists whereby activation of MAP kinases through Ras results in the uncoupling of the Sos/Grb2 complex from tyrosine kinase substrates without blocking the interaction of Sos with Grb2.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Regulação para Baixo , Proteínas Fúngicas/metabolismo , Genes ras , Proteínas Quinases Ativadas por Mitógeno , Proteínas Repressoras/metabolismo , Células Cultivadas , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2 , Humanos , Proteína Quinase 3 Ativada por Mitógeno , Ésteres de Forbol/farmacologia , Fosforilação , Ligação Proteica , Proteínas/metabolismo , Proteína SOS1 , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
11.
Orv Hetil ; 135(47): 2579-84, 1994 Nov 20.
Artigo em Húngaro | MEDLINE | ID: mdl-7824256

RESUMO

Growth factors, such as insulin, EGF, PDGF, are indispensable elements of the regulation of cell proliferation. In the last years signal transduction pathways of many growth factors have been elucidated. p21 ras, a proto-oncogene product, plays a central role in growth signalling. The medical importance of the investigation of Ras proteins comes from the fact that mutant forms of Ras genes were found in a number of human tumours. The review summarizes the signal transduction pathways of growth factors, with special respect to the connection of protooncogenes, tumour suppressor genes and cancer research.


Assuntos
Genes Supressores de Tumor , Neoplasias/genética , Oncogenes , Genes Reguladores , Genes ras , Substâncias de Crescimento/genética , Humanos , Insulina/fisiologia , Neoplasias/enzimologia , Neoplasias/fisiopatologia , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo
12.
J Biol Chem ; 269(19): 14081-7, 1994 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-8188688

RESUMO

In T lymphocytes activated via the T cell antigen receptor (TCR), the SH2- and SH3-containing adapter molecule Grb2 forms a complex with the Ras guanine nucleotide exchange protein Sos and tyrosine phospho-proteins. The interaction of Sos with Grb2 is mediated via the Grb2 SH3 domains. In this study, it is shown that a 75-kDa protein is also complexed with the Grb2 SH3 domains in T cells, but not in Rat-1 fibroblasts. The identity of the p75 protein is not known, but immunoblot analysis with phosphotyrosine antibodies indicated that it is rapidly tyrosine-phosphorylated in TCR-activated T cells. This characteristic clearly distinguishes p75 from Sos since Sos is not a phosphotyrosine protein. In vitro binding studies indicated that the p75 phosphotyrosine protein binds to a glutathione S-transferase fusion protein of intact Grb2, but not to a Grb2 fusion protein mutated in its SH3 domains. p75 can also bind to the single COOH-terminal Grb2 SH3 domain, whereas Sos has an in vitro binding preference for the NH2-terminal Grb2 SH3 domain. Collectively, these data indicate that in T cells, two proteins can complex with the Grb2 SH3 domains: Sos and a p75 molecule that is tyrosine-phosphorylated in TCR-activated cells. The significance of p75 association with Grb2 is not clear, but by analogy with Sos, p75 is a potential candidate for a Grb2 effector protein. Data are presented showing that the interaction of the Grb2 SH2 domains with tyrosine phosphoproteins may be regulated by conformational restraints imposed by different molecules complexing with the Grb2 SH3 domains. It is thus possible to speculate that the interaction of either p75 or Sos with the Grb2 SH3 domain may influence the interaction of the Grb2 SH2 domain with tyrosine phosphoproteins.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Ativação Enzimática , Receptores ErbB/química , Proteína Adaptadora GRB2 , Nucleotídeos de Guanina/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas/química , Ratos , Proteínas Son Of Sevenless , Especificidade por Substrato
13.
Mol Cell Biol ; 14(3): 1575-81, 1994 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8114695

RESUMO

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-nSOS with Shc may be an important event in insulin-induced, mSOS-mediated activation of p21ras.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Receptores ErbB/fisiologia , Proteínas/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Proteína Adaptadora GRB2 , Nucleotídeos de Guanina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteínas Son Of Sevenless , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Proteínas Virais/metabolismo
14.
J Biol Chem ; 269(12): 9019-23, 1994 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-7510700

RESUMO

T lymphocytes contain both Grb2, an SH2 and SH3 domain containing adaptor protein, and Sos, a guanine nucleotide exchange factor for Ras. Immunoprecipitates of Sos from the lysates of T cells contain a 36-kDa protein which is phosphorylated on tyrosine residues in response to T cell receptor/CD3 cross-linking. In vitro studies using different bacterially synthesized GST-Sos fusion proteins confirm the formation of complexes containing p36 and the proline-rich COOH-terminal domain of Sos. The use of mutant GST-Grb2 proteins in which both SH3 domains have been mutationally inactivated shows that Grb2 binds to tyrosine phosphorylated p36 via its SH2 domain. In Jurkat cells phosphorylated p36 is localized exclusively in the particulate fraction. In addition, another SH2 domain-containing protein, p52Shc is tyrosine phosphorylated upon TCR.CD3 cross-linking and associates with a 150-kDa phosphotyrosine containing protein. Taken together these data suggest that activation of Ras in T cells via the TCR.CD3 complex might be controlled, at least in part, by mechanisms similar to those found in fibroblasts, involving in this case formation of a complex of Grb2, Sos, and a membrane-bound tyrosine phosphoprotein of molecular mass 36-kDa.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ativação Linfocitária , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tirosina/análogos & derivados , Proteína Adaptadora GRB2 , Proteínas de Ligação ao GTP/metabolismo , Humanos , Substâncias Macromoleculares , Fosfotirosina , Testes de Precipitina , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Son Of Sevenless , Células Tumorais Cultivadas , Tirosina/metabolismo
15.
Biochim Biophys Acta ; 1220(3): 315-22, 1994 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-8305505

RESUMO

We previously observed a 38 kDa protein that was a major protein component of the cytosolic extract of pig granulocytes and the dominant substrate of protein kinase C at supra-physiological Ca2+ concentrations. The purified 38 kDa protein itself required Ca2+ to be phosphorylated by protein kinase C. Now we demonstrate that this protein, which is also present in human granulocytes, is identical to lipocortin I. The identification is based on the chromatographic properties and immunoblot of the purified protein which is also a good substrate for tissue transglutaminase. Phosphorylation of lipocortin I by protein kinase C was investigated in granulocytes permeabilized with streptolysin-O. At physiological intracellular Ca2+ concentrations lipocortin I was not phosphorylated at all. At supra-physiological Ca2+ concentrations (0.5 mM), lipocortin I was also not phosphorylated when protein kinase C was translocated to the membrane by treatment of the cells with phorbol myristate acetate. Its phosphorylation was detectable only in control experiments when protein kinase C was activated in the cytosol by the addition of dioleoylglycerol and phosphatidylserine to the permeabilized cells. The data presented show that, in permeabilized granulocytes, Ca(2+)-lipocortin is not formed at physiological Ca2+ concentrations, and at supra-physiological Ca2+ concentrations the Ca(2+)-lipocortin I is not accessible to protein kinase C bound to the cytoplasmic surface of the plasma membrane.


Assuntos
Anexina A1/sangue , Proteínas de Membrana/metabolismo , Proteína Quinase C/sangue , Animais , Anexina A1/isolamento & purificação , Proteínas de Bactérias , Cálcio/sangue , Membrana Celular/enzimologia , Permeabilidade da Membrana Celular , Citosol/enzimologia , Diglicerídeos/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Isoenzimas/sangue , Proteínas de Membrana/isolamento & purificação , Peso Molecular , Fosfatidilserinas/farmacologia , Fosfoproteínas/sangue , Fosfoproteínas/isolamento & purificação , Fosforilação , Estreptolisinas , Suínos , Acetato de Tetradecanoilforbol/farmacologia
16.
Cell ; 73(3): 611-20, 1993 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-8490966

RESUMO

Antisera against murine Son of sevenless (Sos) recognize a protein of M(r) 155,000 in rat-1 fibroblasts with specific guanine nucleotide exchange activity toward p21c-Ha-ras. Epidermal growth factor (EGF) receptor coimmunoprecipitates with Sos from EGF-stimulated, but not quiescent, cells. The SH2 and SH3 domain-containing "adapter" protein Grb2 is also found in Sos immunoprecipitates in an EGF-inducible manner. In vitro reconstitution shows that Grb2 is required for the binding of activated EGF receptor to Sos. A phosphopeptide corresponding to tyrosine 1068 of the EGF receptor blocks both the assembly of the complex and EGF stimulation of nucleotide exchange on p21ras in a permeabilized cell system. These results suggest that EGF-induced activation of nucleotide exchange on p21ras proceeds through the recruitment of cytosolic Sos to a complex with EGF receptor and Grb2 at the plasma membrane.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Anticorpos , Carcinoma de Células Escamosas , Linhagem Celular , Membrana Celular/metabolismo , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/isolamento & purificação , Fibroblastos/metabolismo , Proteína Adaptadora GRB2 , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Soros Imunes , Immunoblotting , Cinética , Proteínas de Membrana/imunologia , Proteínas de Membrana/isolamento & purificação , Proteínas/genética , Proteínas/isolamento & purificação , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Son Of Sevenless , Células Tumorais Cultivadas
17.
Nature ; 363(6424): 45-51, 1993 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-8479536

RESUMO

The proteins Grb2-Sem-5, Shc and Sos have been implicated in the signalling pathway from tyrosine kinase receptors to Ras. Grb2-Sem-5 binds directly to murine Sos1, a Ras exchange factor, through two SH3 domains. Sos is also associated with ligand-activated tyrosine kinase receptors which bind Grb2-Sem-5, and with the Grb2-Sem-5 binding protein, Shc. Ectopic expression of Drosophila Sos stimulates morphological transformation of rodent fibroblasts. These data define a pathway by which tyrosine kinases act through Ras to control cell growth and differentiation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Diferenciação Celular , Proteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans , Células Cultivadas , Clonagem Molecular , DNA de Cadeia Simples , Drosophila , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2 , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Ratos , Proteínas Son Of Sevenless
18.
Mol Cell Biol ; 13(3): 1903-10, 1993 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8441421

RESUMO

Treatment of intact Rat-1 fibroblasts with epidermal growth factor (EGF) leads to rapid activation of cellular ras-encoded proteins. By using the bacterial toxin streptolysin O to permeabilize these cells, it was shown that the low basal rate at which guanine nucleotides bind to, and dissociate from, ras-encoded protein in quiescent fibroblasts was greatly accelerated by EGF treatment. Nucleotide binding to other proteins was not affected. Stimulation of nucleotide exchange on ras-encoded protein required tyrosine kinase but not phospholipase activity. EGF had no effect on total GTPase-activating protein activity. Regulation of ras-encoded protein in Rat-1 fibroblasts is therefore mediated by stimulation, either directly or indirectly, of ras-encoded protein-specific guanine nucleotide exchange factors by the EGF receptor tyrosine kinase.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Fibroblastos/metabolismo , Nucleotídeos de Guanina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Proteínas de Bactérias , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Proteínas Ativadoras de GTPase , Genisteína , Isoflavonas/farmacologia , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/efeitos dos fármacos , Ratos , Estreptolisinas/farmacologia , Proteínas Ativadoras de ras GTPase
19.
Biochem Biophys Res Commun ; 187(2): 730-6, 1992 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-1530631

RESUMO

Purified type II (beta) and type III (alpha) protein kinase C phosphorylates highly purified polyADP-ribose polymerase in vitro whereby 2 mols of phosphate are transferred from ATP to serine and threonine residues present in the 36 and 56 kDa polypeptide domains of the polymerase protein. Calf thymus DNA was a non-competitive inhibitor of the protein kinase C catalyzed phosphorylation of polyADP-ribose polymerase. Coincidental with the phosphorylation of the protein the polymerase activity and DNA binding capacity of polyADP-ribose polymerase were inhibited. These in vitro findings may have possible cell biological significance in cellular signal transduction.


Assuntos
DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Quinase C/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Ativação Enzimática , Isoenzimas/metabolismo , Fosforilação , Coelhos
20.
Int J Biochem ; 24(5): 777-82, 1992 May.
Artigo em Inglês | MEDLINE | ID: mdl-1592153

RESUMO

1. The oligopeptide AAASFKAKK which contains recognition motifs similar to that found in the surrounding of the site of H1 histone phosphorylated by protein kinase C is unable to compete with H1 histone for the type II and type III isoenzymes, though it is a good substrate for protein kinase C and it is able to compete with a physiological substrate of the enzyme. 2. Among several oligopeptides tested as an alternative substrate a very basic peptide proved to be the most effective inhibitor of H1 histone phosphorylation. This oligopeptide substrate contains basic recognition motifs at both sides of the phosphorylated residue at variance with the sequence of H1 histone in the surrounding of the phosphorylated site.


Assuntos
Histonas/metabolismo , Isoenzimas/metabolismo , Oligopeptídeos/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Oligopeptídeos/química , Fosforilação , Proteína Quinase C/química , Especificidade por Substrato
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