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1.
Int J Mol Sci ; 22(24)2021 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-34947971

RESUMO

Crkl is a protein involved in the onset of several cancer pathologies that exerts its function only through its protein-protein interaction domains, a SH2 domain and two SH3 domains. SH3 domains are small protein interaction modules that mediate the binding and recognition of proline-rich sequences. One of the main physiological interactors of Crkl is C3G (also known as RAPGEF1), an interaction with key implications in regulating cellular growth and differentiation, cell morphogenesis and adhesion processes. Thus, understanding the interaction between Crkl and C3G is fundamental to gaining information about the molecular determinants of the several cancer pathologies in which these proteins are involved. In this paper, through a combination of fast kinetics at different experimental conditions and site-directed mutagenesis, we characterize the binding reaction between the N-SH3 domain of Crkl and a peptide mimicking a specific portion of C3G. Our results show a clear effect of pH on the stability of the complex, due to the protonation of negatively charged residues in the binding pocket of N-SH3. Our results are discussed under the light of previous work on SH3 domains.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Mutagênese Sítio-Dirigida/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Sítios de Ligação , Adesão Celular , Diferenciação Celular , Proliferação de Células , Fator 2 de Liberação do Nucleotídeo Guanina/química , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Eletricidade Estática
2.
Biochim Biophys Acta Mol Cell Res ; 1868(5): 118964, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33450305

RESUMO

GSK3ß, a ubiquitously expressed Ser/Thr kinase, regulates cell metabolism, proliferation and differentiation. Its activity is spatially and temporally regulated dependent on external stimuli and interacting partners, and its deregulation is associated with various human disorders. In this study, we identify C3G (RapGEF1), a protein essential for mammalian embryonic development as an interacting partner and substrate of GSK3ß. In vivo and in vitro interaction assays demonstrated that GSK3ß and Akt are present in complex with C3G. Molecular modelling and mutational analysis identified a domain in C3G that aids interaction with GSK3ß, and overlaps with its nuclear export sequence. GSK3ß phosphorylates C3G on primed as well as unprimed sites, and regulates its subcellular localization. Over-expression of C3G resulted in activation of Akt and inactivation of GSK3ß. Huntingtin aggregate formation, dependent on GSK3ß inhibition, was enhanced upon C3G overexpression. Stable clones of C2C12 cells generated by CRISPR/Cas9 mediated knockdown of C3G, that cannot differentiate, show reduced Akt activity and S9-GSK3ß phosphorylation compared to wild type cells. Co-expression of catalytically active GSK3ß inhibited C3G induced myocyte differentiation. C3G mutant defective for GSK3ß phosphorylation, does not alter S9-GSK3ß phosphorylation and, is compromised for inducing myocyte differentiation. Our results show complex formation and reciprocal regulation between GSK3ß and C3G. We have identified a novel function of C3G as a negative regulator of GSK3ß, a property important for its ability to induce myogenic differentiation.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/química , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Mutação , Mioblastos/citologia , Animais , Células COS , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Regulação da Expressão Gênica , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Camundongos , Desenvolvimento Muscular , Mioblastos/metabolismo , Fosforilação
3.
J Mol Biol ; 427(24): 3908-20, 2015 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-26456136

RESUMO

The signal adapter protein c-CrkII from chicken but not from human uses isomerization at Pro238 in the SH3C domain to regulate the activity of the SH3N domain. The different behavior of human and chicken c-CrkII originates from only two differences in sequence, at positions 239 after Pro238 and 272 in the N-Src loop of SH3C. We analyzed the kinetics of substrate binding to SH3N and an assay for its coupling with Pro238 isomerization in SH3C to identify the molecular path from Pro238 to the substrate binding site of SH3N. The trans→cis isomerization at Pro238 and a relocation of Phe239 re-organize the energetics of a hydrophobic cluster in the N-Src loop of SH3C and re-shape this region to optimize its interactions with SH3N. Concomitantly, the backbone becomes strained at Met272. We suggest that, in human c-CrkII, movement at position 239 and strain at position 272 are not tolerated because the ß-branched residues Ile239 and Val272 restrain the backbone mobility and thus destabilize the cis Pro238 form.


Assuntos
Prolina/química , Proteínas Proto-Oncogênicas c-crk/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Galinhas , Fator 2 de Liberação do Nucleotídeo Guanina/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termodinâmica
4.
PLoS One ; 6(8): e23681, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21876762

RESUMO

The guanine nucleotide exchange factor, C3G (RapGEF1), functions in multiple signaling pathways involved in cell adhesion, proliferation, apoptosis and actin reorganization. C3G is regulated by tyrosine phosphorylation on Y504, known to be mediated by c-Abl and Src family kinases. In the present study we explored the possibility of cellular phospho-C3G (pC3G) being a substrate of the intracellular T-cell protein tyrosine phosphatase TC-PTP (PTPN2) using the human neuroblastoma cell line, IMR-32. In vivo and in vitro binding assays demonstrated interaction between C3G and TC-PTP. Interaction is mediated through the Crk-binding region of C3G and C-terminal noncatalytic residues of TC-PTP. C3G interacted better with a substrate trap mutant of TC48 and this complex formation was inhibited by vanadate. Endogenous pC3G colocalized with catalytically inactive mutant TC48 in the Golgi. Expression of TC48 abrogated pervanadate and c-Src induced phosphorylation of C3G without affecting total cellular phospho-tyrosine. Insulin-like growth factor treatment of c-Src expressing cells resulted in dephosphorylation of C3G dependent on the activity of endogenous TC48. TC48 expression inhibited forskolin induced tyrosine phosphorylation of C3G and neurite outgrowth in IMR-32 cells. Our results identify a novel Golgi localized substrate of TC48 and delineate a role for TC48 in dephosphorylation of substrates required during differentiation of human neuroblastoma cells.


Assuntos
Diferenciação Celular , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colforsina/farmacologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/química , Células HEK293 , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 2/química , Relação Estrutura-Atividade , Vanadatos/farmacologia , Quinases da Família src/metabolismo
5.
Biosci Rep ; 31(4): 231-44, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21366540

RESUMO

C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) is a ubiquitously expressed member of a class of molecules called GEFs (guanine-nucleotide-exchange factor) that activate small GTPases and is involved in pathways triggered by a variety of signals. It is essential for mammalian embryonic development and many cellular functions in adult tissues. C3G participates in regulating functions that require cytoskeletal remodelling such as adhesion, migration, maintenance of cell junctions, neurite growth and vesicle traffic. C3G is spatially and temporally regulated to act on Ras family GTPases Rap1, Rap2, R-Ras, TC21 and Rho family member TC10. Increased C3G protein levels are associated with differentiation of various cell types, indicating an important role for C3G in cellular differentiation. In signalling pathways, C3G serves functions dependent on catalytic activity as well as protein interaction and can therefore integrate signals necessary for the execution of more than one cellular function. This review summarizes our current knowledge of the biology of C3G with emphasis on its role as a transducer of signals to the actin cytoskeleton. Deregulated C3G may also contribute to pathogenesis of human disorders and therefore could be a potential therapeutic target.


Assuntos
Actinas/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Transdução de Sinais , Animais , Apoptose , Proliferação de Células , Sobrevivência Celular , Citoesqueleto/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/química , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Humanos , Modelos Biológicos , Proteínas Monoméricas de Ligação ao GTP/metabolismo
6.
Oncogene ; 29(32): 4528-42, 2010 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-20581864

RESUMO

The c-Abl tyrosine kinase maintains cellular homeostasis through its ability to regulate apoptosis and actin dynamics. In vivo, c-Abl activity is stringently regulated and mechanisms involved are not fully understood. Here, we identified the Rap1 guanine nucleotide exchange factor, C3G (RapGEF1), as a substrate and an effector of c-Abl-mediated functions. Ectopic expression of c-Abl in mammalian cell lines, known to induce apoptosis, resulted in phosphorylation of endogenous C3G on Y504 coincident with cell detachment and chromatin condensation. Phosphorylation of C3G coincided with restricted c-Abl activation in regions rich in actin, and was dependent on cellular F-actin dynamics. Unlike C3G or c-Abl, p-C3G was resistant to detergent extraction, suggesting its enhanced affinity for the cytoskeleton. Localized C3G phosphorylation and coincidence with cells undergoing cell death was dependent on F-actin-binding domain (FABD) of c-Abl. Activation of endogenous c-Abl by oxidative stress was associated with phosphorylation of cellular C3G on Y504. Inhibition of C3G expression and function using RNAi or dominant-negative approaches inhibited c-Abl-mediated cell death. These findings identify C3G as a novel target of c-Abl and also show that FABD of c-Abl is essential for regulation of its restricted activation to induce apoptosis.


Assuntos
Actinas/metabolismo , Apoptose , Fator 2 de Liberação do Nucleotídeo Guanina/química , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/metabolismo , Anticorpos/imunologia , Anticorpos/metabolismo , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Fator 2 de Liberação do Nucleotídeo Guanina/imunologia , Humanos , Espaço Intracelular/metabolismo , Estresse Oxidativo , Fosforilação , Estrutura Terciária de Proteína , Transporte Proteico , Tirosina/metabolismo
7.
Genetics ; 177(4): 2445-56, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17947423

RESUMO

In Drosophila melanogaster oocytes, the C(3)G protein comprises the transverse filaments (TFs) of the synaptonemal complex (SC). Like other TF proteins, such as Zip1p in yeast and SCP1 in mammals, C(3)G is composed of a central coiled-coil-rich domain flanked by N- and C-terminal globular domains. Here, we analyze in-frame deletions within the N- and C-terminal regions of C(3)G in Drosophila oocytes. As is the case for Zip1p, a C-terminal deletion of C(3)G fails to attach to the lateral elements of the SC. Instead, this C-terminal deletion protein forms a large cylindrical polycomplex structure. EM analysis of this structure reveals a polycomplex of concentric rings alternating dark and light bands. However, unlike both yeast and mammals, all three proteins deleted for N-terminal regions completely abolished both SC and polycomplex formation. Both the N- and C-terminal deletions significantly reduce or abolish meiotic recombination similarly to c(3)G null homozygotes. To explain these data, we propose that in Drosophila the N terminus, but not the C-terminal globular domain, of C(3)G is critical for the formation of antiparallel pairs of C(3)G homodimers that span the central region and thus for assembly of complete TFs, while the C terminus is required to affix these homodimers to the lateral elements.


Assuntos
Pareamento Cromossômico , Proteínas de Drosophila/fisiologia , Fator 2 de Liberação do Nucleotídeo Guanina/fisiologia , Recombinação Genética , Animais , Dimerização , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Fator 2 de Liberação do Nucleotídeo Guanina/química , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Meiose , Microscopia Eletrônica , Oócitos/química , Oócitos/ultraestrutura , Estrutura Terciária de Proteína , Deleção de Sequência
8.
BMC Cell Biol ; 5: 31, 2004 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-15320955

RESUMO

BACKGROUND: The guanine nucleotide exchange factor C3G (RapGEF1) along with its effector proteins participates in signaling pathways that regulate eukaryotic cell proliferation, adhesion, apoptosis and embryonic development. It activates Rap1, Rap2 and R-Ras members of the Ras family of GTPases. C3G is activated upon phosphorylation at tyrosine 504 and therefore, determining the localization of phosphorylated C3G would provide an insight into its site of action in the cellular context. RESULTS: C3G is phosphorylated in vivo on Y504 upon coexpression with Src or Hck, two members of the Src family tyrosine kinases. Here we have determined the subcellular localization of this protein using antibodies specific to C3G and Tyr 504 phosphorylated C3G (pY504 C3G). While exogenously expressed C3G was present mostly in the cytosol, pY504 C3G formed upon Hck or Src coexpression localized predominantly at the cell membrane and the Golgi complex. Tyrosine 504-phosphorylated C3G showed colocalization with Hck and Src. Treatment of Hck and C3G transfected cells with pervanadate showed an increase in the cytosolic staining of pY504 C3G suggesting that tyrosine phosphatases may be involved in dephosphorylating cytosolic phospho-C3G. Expression of Src family kinases or treatment of cells with pervanadate resulted in an increase in endogenous pY504 C3G, which was localized predominantly at the Golgi and the cell periphery. Endogenous pY504 C3G at the cell periphery colocalized with F-actin suggesting its presence at the subcortical actin cytoskeleton. Disruption of actin cytoskeleton by cytochalasin D abolished phospho-C3G staining at the periphery of the cell without affecting its Golgi localization. CONCLUSIONS: These findings show that tyrosine kinases involved in phosphorylation of C3G are responsible for regulation of its localization in a cellular context. We have demonstrated the localization of endogenous C3G modified by tyrosine phosphorylation to defined subcellular domains where it may be responsible for restricted activation of signaling pathways.


Assuntos
Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Vanadatos/farmacologia , Animais , Células COS , Chlorocebus aethiops , Citosol/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/química , Células HeLa , Humanos , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Fosfotirosina/análise , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-hck , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Frações Subcelulares/metabolismo
9.
Anal Chem ; 75(22): 6163-70, 2003 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-14615996

RESUMO

A new chip-based method to identify protein-protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by microLC-MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC-MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein-protein interactions.


Assuntos
Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Espectrometria de Massas/métodos , Análise Serial de Proteínas/métodos , Calmodulina/química , Calmodulina/metabolismo , Cromatografia Líquida , Fluorescência , Fator 2 de Liberação do Nucleotídeo Guanina/química , Espectrometria de Massas/instrumentação , Testes de Precipitina , Ligação Proteica , Tripsina/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo
10.
J Biol Chem ; 278(52): 52188-94, 2003 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-14551197

RESUMO

The hematopoietic cell kinase Hck is a Src family tyrosine kinase expressed in cells of myelomonocytic lineage, B lymphocytes, and embryonic stem cells. To study its role in signaling pathways we used the Hck-SH3 domain in protein interaction cloning and identified C3G, the guanine nucleotide exchange factor for Rap1 and R-Ras, as a protein that associated with Hck. This interaction was direct and was mediated partly through the proline-rich region of C3G. C3G could be co-immunoprecipitated with Hck from Cos-1 cells transfected with Hck and C3G. C3G was phosphorylated on tyrosine 504 in cells when coexpressed with Hck but not with a catalytically inactive mutant of Hck. Phosphorylation of endogenous C3G at Tyr-504 was increased by treatment of human myelomonocytic THP-1 cells with mercuric chloride, which is known to activate Hck tyrosine kinase specifically. Coexpression of Hck with C3G induced a high level of apoptosis in many cell lines by 30-42 h of transfection. Induction of apoptosis was not dependent on Tyr-504 phosphorylation or the catalytic domain of C3G but required the catalytic activity of Hck. Using dominant negative constructs of caspases we found that caspase-1, -8, and -9 are involved in this apoptotic pathway. These results suggest that C3G and Hck interact physically and functionally in vivo to activate kinase-dependent and caspase-mediated apoptosis, which is independent of catalytic domain of C3G.


Assuntos
Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Apoptose , Western Blotting , Células COS , Caspase 1/metabolismo , Caspase 8 , Caspase 9 , Caspases/metabolismo , Catálise , Domínio Catalítico , Linhagem Celular , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Genes Dominantes , Vetores Genéticos , Glutationa Transferase/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/química , Células HeLa , Humanos , Cloreto de Mercúrio/farmacologia , Microscopia de Fluorescência , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteínas Proto-Oncogênicas c-hck , Transdução de Sinais , Fatores de Tempo , Transfecção , Tirosina/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Domínios de Homologia de src
11.
J Biol Chem ; 278(29): 27301-11, 2003 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-12734187

RESUMO

We demonstrate here that growth hormone (GH) stimulates the activation of Rap1 and Rap2 in NIH-3T3 cells. Full activation of Rap1 and Rap2 by GH necessitated the combined activity of both JAK2 and c-Src kinases, although c-Src was predominantly required. GH-stimulated Rap1 and Rap2 activity was also demonstrated to be CrkII-C3G-dependent. GH stimulated the tyrosine phosphorylation of C3G, which again required the combined activity of JAK2 and c-Src. C3G tyrosine residue 504 was required for GH-stimulated Rap activation. Activated Rap1 inhibited GH-stimulated activation of RalA and subsequent GH-stimulated p44/42 MAP kinase activity and Elk-1-mediated transcription. In addition, we demonstrated that C3G-Rap1 mediated CrkII enhancement of GH-stimulated JNK/SAPK activity. We have therefore identified a linear JAK2-independent pathway switching GH-stimulated p44/42 MAP kinase and JNK/SAPK activities.


Assuntos
Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Hormônio do Crescimento Humano/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Células 3T3 , Animais , Proteína Tirosina Quinase CSK , Fator 2 de Liberação do Nucleotídeo Guanina/química , Humanos , Janus Quinase 2 , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-crk , Proteínas Recombinantes/farmacologia , Tirosina/química , Proteínas rap de Ligação ao GTP/metabolismo , Quinases da Família src
12.
Biochem Biophys Res Commun ; 286(1): 61-6, 2001 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-11485308

RESUMO

Two types of C3G cDNA were isolated from mouse 3T3-L1 adipocyte cDNA library. A 114-bp sequence in the middle of C3G cDNA is deleted in the short type cDNA. By RT-PCR analysis, it was found that these two types of C3G mRNA existed in all the mouse tissues. Sequence comparison revealed 88% nucleotide sequence identity between mouse and human C3G cDNA. Comparison of mouse C3G cDNA with the human genome database suggested that this 114-bp sequence comprised an entire exon, and it is confirmed by PCR analysis using mouse genomic DNA and cDNA template. These results indicate that two C3G mRNAs and proteins result from alternative RNA splicing.


Assuntos
Processamento Alternativo , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Fator 2 de Liberação do Nucleotídeo Guanina/química , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
13.
J Biol Chem ; 275(17): 13118-25, 2000 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-10777617

RESUMO

In studies to define mechanisms of ERK activation in Chinese hamster ovary cells, we have observed an inverse correlation between CRKII-C3G complex formation and ERK activity. That is, we were able to coprecipitate the guanine nucleotide exchange factor C3G with the adaptor protein CRKII in lysates from suspended cells that had low ERK activity, but we could not do so or could do so less efficiently in lysates of adherent cells with increased ERK activity. Consistent with the presence of a functional CRKII-C3G complex, we detected more GTP-loaded RAP1 in suspension than adherent lysates. Overexpression of cDNAs encoding B-RAF, CRKII W109L, and PTP1B C215S activated ERK in suspension cells, the latter two constructs also disrupting CRKII-C3G complex formation. Finally, we have also observed that certain integrin alpha subunit cytoplasmic splice variants differentially regulate ERK1/2 but also in a manner that correlated with levels of a CRKII-C3G complex. Thus, these data suggest the involvement of integrins in an ERK suppression pathway mediated by CRKII-C3G complex formation and downstream signaling from activated RAP1.


Assuntos
Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Integrinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas , Processamento Alternativo , Sequência de Aminoácidos , Animais , Western Blotting , Células CHO , Adesão Celular , Cricetinae , Citometria de Fluxo , Fator 2 de Liberação do Nucleotídeo Guanina/química , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Complexo Antigênico da Nefrite de Heymann , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas c-crk , Homologia de Sequência de Aminoácidos , Proteínas Ativadoras de ras GTPase/metabolismo
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