RESUMO
The SOS family of Ras-GEFs encompasses two highly homologous and widely expressed members, SOS1 and SOS2. Despite their similar structures and expression patterns, early studies of constitutive KO mice showing that SOS1-KO mutants were embryonic lethal while SOS2-KO mice were viable led to initially viewing SOS1 as the main Ras-GEF linking external stimuli to downstream RAS signaling, while obviating the functional significance of SOS2. Subsequently, different genetic and/or pharmacological ablation tools defined more precisely the functional specificity/redundancy of the SOS1/2 GEFs. Interestingly, the defective phenotypes observed in concomitantly ablated SOS1/2-DKO contexts are frequently much stronger than in single SOS1-KO scenarios and undetectable in single SOS2-KO cells, demonstrating functional redundancy between them and suggesting an ancillary role of SOS2 in the absence of SOS1. Preferential SOS1 role was also demonstrated in different RASopathies and tumors. Conversely, specific SOS2 functions, including a critical role in regulation of the RAS-PI3K/AKT signaling axis in keratinocytes and KRAS-driven tumor lines or in control of epidermal stem cell homeostasis, were also reported. Specific SOS2 mutations were also identified in some RASopathies and cancer forms. The relevance/specificity of the newly uncovered functional roles suggests that SOS2 should join SOS1 for consideration as a relevant biomarker/therapy target.
Assuntos
Proteína SOS1/fisiologia , Proteínas Son Of Sevenless/fisiologia , Animais , Humanos , Neoplasias/metabolismoRESUMO
Son of sevenless homolog 1 (SOS1) protein is a ubiquitously expressed adapter. As a key protein in intracellular signaling, SOS1 plays an important role in many signal transduction pathways, such as Ras and Rac signaling pathways. The abnormal expression or mutation of SOS1 is closely related to clinical diseases. In this article, we review research progress on SOS1 functions and its roles in physiology and pathophysiology.
Assuntos
Proteína SOS1/fisiologia , Transdução de Sinais , Animais , Humanos , MutaçãoRESUMO
SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.
Assuntos
Proteína SOS1/fisiologia , Proteínas ras/metabolismo , Regulação Alostérica , Animais , Membrana Celular/metabolismo , Galinhas , Endocitose , Ativação Enzimática , Humanos , Células Jurkat , Cinética , Bicamadas Lipídicas/química , Sistema de Sinalização das MAP Quinases , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Proteínas Proto-Oncogênicas c-bcr/fisiologia , Proteína SOS1/química , Proteínas ras/químicaRESUMO
Using a 4-hydroxytamoxifen (4OHT)-inducible, conditional Sos1-null mutation, we analyzed wild-type (WT), single Sos1-KO, Sos2-KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEF) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2-KO counterparts. The Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation, in comparison with WT and Sos2-KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Cellular proliferation and migration were impaired in Sos1-KO and Sos1/2-DKO MEFs in comparison with WT and Sos2-KO MEFs, whereas cell adhesion was only impaired upon depletion of both Sos isoforms. RasGTP formation was practically absent in Sos1/2-DKO MEFs as compared with the other genotypes and extracellular signal-regulated kinase phosphorylation showed only significant reduction after combined Sos1/2 depletion. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1-KO and the Sos1/2-DKO cells as compared with Sos2-KO and WT MEFs. Interestingly, treatment of the MEF cultures with antioxidants corrected the altered phenotypes of Sos1-KO and Sos1/2-DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2-KO MEFs. Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability.
Assuntos
Proliferação de Células , Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Proteína SOS1/fisiologia , Animais , Antioxidantes/farmacologia , Adesão Celular , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Dano ao DNA , Camundongos , Transdução de Sinais , Proteínas Son Of Sevenless/fisiologiaRESUMO
Son of sevenless 1 (SOS1) is a Ras-specific guanine-nucleotide-exchange factor (GEF) that mediates intracellular signaling processes induced by receptor tyrosine kinases. In this study, we show that CIIA (also known as VPS28) physically associates with SOS1 and thereby inhibits the GEF activity of SOS1 on Ras, which prevents the epidermal growth factor (EGF)-induced activation of the Ras-Erk1/2 pathway. Furthermore, CIIA inhibited cyclin D1 expression, as well as DNA, synthesis in response to EGF. Intriguingly, CIIA failed to inhibit the Ras-specific GEF activity of Noonan-syndrome-associated SOS1 mutants (M269R, R552G, W729L and E846K). Taken together, our results suggest that CIIA functions as a negative modulator of the SOS1-Ras signaling events initiated by peptide growth factors including EGF.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Sistema de Sinalização das MAP Quinases , Proteína SOS1/fisiologia , Proteínas ras/metabolismo , Animais , Ciclina D1/genética , Ciclina D1/metabolismo , Replicação do DNA , Cães , Fator de Crescimento Epidérmico/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Camundongos , Mutação de Sentido Incorreto , Células NIH 3T3 , Síndrome de Noonan/genéticaRESUMO
The activation of the small guanosine triphosphatase Ras by the guanine nucleotide exchange factor (GEF) Sos1 (Son of Sevenless 1) is a central feature of many receptor-stimulated signaling pathways. In developing T cells (thymocytes), Sos1-dependent activation of extracellular signal-regulated kinase (ERK) is required to stimulate cellular proliferation and differentiation. We showed that in addition to its GEF activity, Sos1 acted as a scaffold to nucleate oligomerization of the T cell adaptor protein LAT (linker for activation of T cells) in vivo. The scaffold function of Sos1 depended on its ability to bind to the adaptor protein Grb2. Furthermore, the GEF activity of Sos1 and the Sos1-dependent oligomerization of LAT were separable functions in vivo. Whereas the GEF activity of Sos1 was required for optimal ERK phosphorylation in response to T cell receptor (TCR) stimulation, the Sos1-dependent oligomerization of LAT was required for maximal TCR-dependent phosphorylation and activation of phospholipase C-γ1 and Ca(2+) signaling. Finally, both of these Sos1 functions were required for early thymocyte proliferation. Whereas transgenic restoration of either the GEF activity or the LAT oligomerization functions of Sos1 alone failed to rescue thymocyte development in Sos1-deficient mice, simultaneous reconstitution of these two signals in the same cell restored normal T cell development. This ability of Sos1 to act both as a RasGEF and as a scaffold to nucleate Grb2-dependent adaptor oligomerization may also occur in other Grb2-dependent pathways, such as those activated by growth factor receptors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteína SOS1/genética , Proteína SOS1/fisiologia , Animais , Sinalização do Cálcio , Diferenciação Celular , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Adaptadora GRB2/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Nucleotídeos/química , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Timócitos/citologia , Transgenes , Proteínas ras/metabolismoRESUMO
Hirsutism is the development of androgen-dependent terminal body hair in women in places in which terminal hair are normally not found. It is often associated with hyperandrogenemia and/or polycystic ovary syndrome (PCOS), but the existence of uncommom hirsutism forms that are not related to altered androgen plasma levels lead also to the definition of - idiopathic hirsutism. Although the pathophysiology of hirsutism has been linked to increasing 5-alpha reductase (SRD5A) activity and to an alteration of the androgen receptor (AR) transcriptional machinery, many aspects remain unclear. In particular, the relationships between androgens and local factors are poorly understood. In the present paper, we selected for a genital skin biopsy, 8 women affected with severe hirsutism (Ferriman-Gallway score greater than 25) but with normal plasma androgen levels, with the exception of slightly higher serum 3alpha-diol-glucuronide levels, and 6 healthy controls and analyzed their androgen- and insulin-specific transcriptional profile using a specific custom low density microarray (AndroChip 2, GPL9164). We identified the over-expression of the Son of Sevenless-1 (SOS1) gene in all of the hirsute skin fibroblast primary cell cultures compared to control healthy women. Since SOS1 is a guanine nucleotide exchange factor that couples receptor tyrosine kinases to the RAS signaling pathway that controls cell proliferation and differentiation, we further analyzed SOS1 expression, protein level and RAS signaling activation pathway in an in vitro model (NHDF, normal human dermal fibroblast cell line). NHDF treated for 24 h with different concentrations of DHT and T showed an increase in SOS1 levels (both mRNA and protein) and also an activation of the RAS pathway. Our in vivo and in vitro data represent a novel preliminary observation that factors activating SOS1 could act as local proliferative modulators linked to the androgen pathway in the pilosebaceous unit. SOS1 over-expression may play a role in the regulation of the RAS/mitogen-activated protein kinase pathway in the skin, in the hair follicle proliferation and cell cycle, suggesting new perspectives in understanding the pathogenesis of idiopathic hirsutism.
Assuntos
Fibroblastos/metabolismo , Hirsutismo/etiologia , Proteína SOS1/fisiologia , Transdução de Sinais/fisiologia , Proteínas ras/fisiologia , Adulto , Células Cultivadas , Di-Hidrotestosterona/farmacologia , Feminino , Genitália Feminina/citologia , Genitália Feminina/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , Proteína SOS1/genética , Testosterona/farmacologiaRESUMO
The v-Crk oncogene product consists of two protein interaction modules, a Src homology 2 (SH2) domain and a Src homology 3 (SH3) domain. Overexpression of CrkI, the cellular homolog of v-Crk, transforms mouse fibroblasts, and elevated CrkI expression is observed in several human cancers. The SH2 and SH3 domains of Crk are required for transformation, but the identity of the critical cellular binding partners is not known. A number of candidate Crk SH3-binding proteins have been identified, including the nonreceptor tyrosine kinases c-Abl and Arg, and the guanine nucleotide exchange proteins C3G, SOS1 and DOCK180. The aim of this study is to determine which of these are required for transformation by CrkI. We found that short hairpin RNA-mediated knockdown of C3G or SOS1 suppressed anchorage-independent growth of NIH-3T3 cells overexpressing CrkI, whereas knockdown of SOS1 alone was sufficient to suppress tumor formation by these cells in nude mice. Knockdown of C3G was sufficient to revert morphological changes induced by CrkI expression. By contrast, knockdown of Abl family kinases or their inhibition with imatinib enhanced anchorage-independent growth and tumorigenesis induced by Crk. These results show that SOS1 is essential for CrkI-induced fibroblast transformation, and also reveal a surprising negative role for Abl kinases in Crk transformation.
Assuntos
Transformação Celular Neoplásica , Proteínas Proto-Oncogênicas c-crk/fisiologia , Domínios de Homologia de src , Animais , Apoptose , Fator 2 de Liberação do Nucleotídeo Guanina/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Nus , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/fisiologia , Proteínas Proto-Oncogênicas c-crk/química , Proteína SOS1/fisiologia , Transdução de SinaisRESUMO
EGFRvIII, a frequent genetic alteration of the epidermal growth factor receptor (EGFR), has been shown to increase the migratory potential of tumor cells and normal fibroblasts. Previously, we showed that signal regulatory protein alpha1 (SIRPalpha1) receptors interact with SHP-2 to inhibit wild-type (wt) EGFR-mediated tumor migration, survival and cell transformation. However, the effects of SIRPalpha1 inhibitory receptors on EGFRvIII-mediated phenotypes are unclear. The aim of this study was to investigate the effect of SIRPalpha1 receptor on the EGFRvIII signalosome and phenotypes. Overexpression of SIRPalpha1 in U87MG.EGFRvIII cells inhibited transformation and migration in a MAPK-dependent manner, and is independent of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. We observed reduced EGFRvIII/SHP-2/Gab1/Grb2/Sos-1 interaction and enhanced SIRP/SHP-2 association in U87MG.EGFRvIII/SIRPalpha1 cells when compared with empty vector control cells. Interestingly, SIRPalpha1 overexpression differentially modulated SHP-2 phosphorylation at tyrosyl 542 and 580 residues, which may regulate Erk1/2 activity and the EGFRvIII phenotype. In addition, SIRPalpha1-expressing cells exhibited reduced focal adhesion kinase (FAK) phosphorylation and its recruitment to the EGFRvIII/Grb2/Sos-1/Gab1/SHP-2 complex. Collectively, our data indicate that SIRPalpha1 specifically affects the SHP-2/FAK/Grb2/Sos-1/MAPK activation loop to downmodulate EGFRvIII-mediated migration and transformation. Further understanding of the molecular interactions between the SIRPalpha1 inhibitory receptor and the EGFRvIII signalosome may facilitate the identification of novel targets to inhibit the EGFRvIII glioblastoma phenotype.
Assuntos
Antígenos de Diferenciação/fisiologia , Transformação Celular Neoplásica , Receptores ErbB/fisiologia , Glioblastoma/patologia , Receptores Imunológicos/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteína Adaptadora GRB2/fisiologia , Humanos , Fenótipo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Proteína SOS1/fisiologiaRESUMO
Suppressor of cytokine signaling 1 (SOCS1) is a recently identified host factor that positively regulates the intracellular trafficking and stability of HIV-1 Gag. We here examine the molecular mechanism by which SOCS1 regulates intercellular Gag trafficking and virus particle production. We find that SOCS1 colocalizes with Gag along the microtubule network and promotes microtubule stability. SOCS1 also increases the amount of Gag associated with microtubules. Both nocodazole treatment and the expression of the microtubule-destabilizing protein, stathmin, inhibit the enhancement of HIV-1 particle production by SOCS1. SOCS1 facilitates Gag ubiquitination and the co-expression of a dominant-negative ubiquitin significantly inhibits the association of Gag with microtubules. We thus propose that the microtubule network plays a role in SOCS1-mediated HIV-1 Gag transport and virus particle formation.
Assuntos
Produtos do Gene gag/fisiologia , HIV-1/fisiologia , Microtúbulos/fisiologia , Proteína SOS1/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Produtos do Gene gag/metabolismo , Humanos , Proteína SOS1/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: Heterozygous gain-of-function mutations in various genes encoding proteins of the Ras-MAPK signalling cascade have been identified as the genetic basis of Noonan syndrome (NS) and cardio-facio-cutaneous syndrome (CFCS). Mutations of SOS1, the gene encoding a guanine nucleotide exchange factor for Ras, have been the most recent discoveries in patients with NS, but this gene has not been studied in patients with CFCS. METHODS AND RESULTS: We investigated SOS1 in a large cohort of patients with disorders of the NS-CFCS spectrum, who had previously tested negative for mutations in PTPN11, KRAS, BRAF, MEK1 and MEK2. Missense mutations of SOS1 were discovered in 28% of patients with NS. In contrast, none of the patients classified as having CFCS was found to carry a pathogenic sequence change in this gene. CONCLUSION: We have confirmed SOS1 as the second major gene for NS. Patients carrying mutations in this gene have a distinctive phenotype with frequent ectodermal anomalies such as keratosis pilaris and curly hair. However, the clinical picture associated with SOS1 mutations is different from that of CFCS. These findings corroborate that, despite being caused by gain-of-function mutations in molecules belonging to the same pathway, NS and CFCS scarcely overlap genotypically.
Assuntos
Cardiopatias/genética , Síndrome de Noonan/genética , Proteína SOS1/genética , Proteína SOS1/fisiologia , Dermatopatias/genética , Síndrome , Sequência de Aminoácidos , Estatura , Constrição Patológica , Feminino , Cardiopatias/congênito , Heterozigoto , Humanos , Masculino , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fatores ras de Troca de Nucleotídeo Guanina/metabolismoRESUMO
The oncoprotein Ras is anchored in lipid membranes due to its C-terminal lipid modification. The ubiquitously expressed Ras nucleotide exchange-factor hSOS1 promotes nucleotide exchange and thus Ras activation. This reaction is enhanced by a positive feedback loop whereby activated Ras binds to an allosteric site of SOS to enhance GEF activity. Here we present biochemical data showing that prenylation of both active site bound and allosterically bound N-Ras is required for efficient hSOS1-promoted nucleotide exchange. Our results indicate that prenyl sensitivity of the allosteric feedback-activation is mediated by the PH domain of hSOS1. Farnesylation of Ras thereby allows hSOS1 to bind even GDP-loaded allosteric regulator to maintain basal hSOS1-activity.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína SOS1/fisiologia , Proteínas ras/metabolismo , Sítio Alostérico , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/fisiologia , Retroalimentação Fisiológica/fisiologia , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Humanos , Fragmentos de Peptídeos/fisiologia , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Ligação Proteica , Prenilação de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteína SOS1/genética , Proteína SOS1/metabolismo , Homologia Estrutural de Proteína , Proteínas ras/fisiologiaRESUMO
Evolutionary conserved members of the Ras superfamily of small GTP-binding proteins function as binary molecular switches to control diverse biological processes. In the context of cellular signaling, these include functions in exocytic and endocytic trafficking, as well as roles in signal relay downstream of various cell surface receptors. We previously reviewed roles played by the large family of GTPase, activating proteins in these processes. In this companion review, we highlight recent findings relating to the regulation of another major class of Ras superfamily regulatory proteins, the guanine nucleotide exchange factors.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Transdução de Sinais/fisiologia , Animais , Doenças Genéticas Inatas/etiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Proteína SOS1/fisiologiaRESUMO
Two important Ras guanine nucleotide exchange factors, Son of sevenless (Sos) and Ras guanine nucleotide releasing protein (RasGRP), have been implicated in controlling Ras activation when cell surface receptors are stimulated. To address the specificity or redundancy of these exchange factors, we have generated Sos1/Sos2 double- or RasGRP3-deficient B cell lines and determined their ability to mediate Ras activation upon B cell receptor (BCR) stimulation. The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2. Furthermore, we show that BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-gamma2-deficient B cells. This defective Ras activation is suppressed by the expression of RasGRP3 as a membrane-attached form, suggesting that phospholipase C-gamma2 regulates RasGRP3 localization and thereby Ras activation.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Fosfolipases Tipo C/fisiologia , Proteínas ras/fisiologia , Sequência de Aminoácidos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fosfolipase C gama , Proteína SOS1/fisiologia , Transdução de Sinais , Proteínas Son Of Sevenless/fisiologia , Fatores ras de Troca de Nucleotídeo GuaninaRESUMO
A 19 kDa protein was identified to associate with the Dbl oncogene homology domain of Sos1 (Sos-DH) and was purified from rat brains by GST-Sos-DH affinity chromatography. Peptide sequencing revealed that the protein is identical to light chain 3 (LC3), a microtubule-associated protein. LC3 coimmunoprecipitated with Sos1, and GST-LC3 was capable of forming complexes with Sos1 in in vitro GST-pull down assay. Furthermore, LC3 was colocalized with Sos1 in cells, as determined by immunohistochemistry. While Sos1 stimulated the guanine nucleotide exchange reaction on Rac1, LC3 suppressed the ability of Sos1 to activate Rac1 in in vitro experiments using COS cell lysates. Consistent with this, overexpression of LC3 decreased the level of active GTP-bound Rac1 in COS cells. Sos1 expression induced membrane ruffling, a downstream target for Rac1, but LC3 expression inhibited this biological effect of Sos1. These findings suggest that LC3 interacts with Sos1 and thereby negatively regulates the Sos1-dependent Rac1 activation leading to membrane ruffling.
Assuntos
Membrana Celular/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Proteína SOS1/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Células 3T3 , Animais , Células COS , Guanosina Difosfato/metabolismo , Camundongos , Subunidades Proteicas , Ratos , Proteína SOS1/químicaRESUMO
The protein hSos1 is a Ras guanine nucleotide exchange factor. In the present study, we investigated the function of the amino-terminal region of the hSos1 protein, corresponding to the first 600 residues, which includes the Dbl and pleckstrin homology (DH and PH) domains. We demonstrated, using a series of truncated mutants, that this region is absolutely necessary for hSos1 activity. Our results suggest that the first 200 residues (upstream of DH domain), which we called the HF motif on the basis of their homology with histone H2A, may exert negative control over the functional activity of the whole hSos1 protein. In vitro binding analysis showed that the HF motif is able to interact specifically with the PH domain of hSos1. The amino-terminal region of hSos1 may be associated in vivo with an expressed HF motif. These findings document the existence of the HF motif located upstream of the DH domain in the hSos1 protein. This motif may be responsible for the negative control of hSos1, probably by intramolecular binding with the PH domain.
Assuntos
Proteína SOS1/química , Proteína SOS1/fisiologia , Células 3T3 , Motivos de Aminoácidos , Animais , Células COS , Linhagem Celular , Deleção de Genes , Genes Reporter , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Modelos Moleculares , Peptídeos , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Son of sevenless (SOS) is a guanine nucleotide exchange factor that activates Ras in response to growth factor stimulation. SOS also appears to serve as a guanine nucleotide exchanger for Rac and, thus, may be involved in cytoskeleton reorganization. Nimnual and Bar-Sagi discuss how these two activities of SOS can be regulated and how SOS may be recruited to different cellular locations through interactions with the adaptor proteins Grb2 and E3b1.
Assuntos
Transdução de Sinais/fisiologia , Proteínas Son Of Sevenless/fisiologia , Animais , Catálise , Ativação Enzimática/fisiologia , Humanos , Proteína SOS1/fisiologia , Proteína Son Of Sevenless de Drosófila/fisiologiaRESUMO
hSos1 isoform II, defined by the presence of a 15 amino acid stretch in its carboxy-terminal region, exhibits higher Grb2 affinity than hSos1 isoform I. In this study, we investigated the cause for this difference and observed that, in addition to the four currently accepted Grb2-binding motifs, a number of additional, putative SH3-minimal binding sites (SH3-MBS) could be identified. The isoform II-specific 15 amino acid stretch contained one of them. Indeed, we demonstrated by site-directed mutagenesis that these SH3-MBS were responsible for the Grb2 interaction, and we found that C-terminal fragments of the two hSos1 isoforms (lacking the four cannonical Grb2-binding motifs, but containing the SH3-minimal binding sites) were able to bind Grb2, with the isoform II fragment showing higher Grb2 affinity than the corresponding isoform I fragment. Furthermore, we provide evidence that C-terminal truncated mutants of either hSos1 isoform, containing only the SH3-minimal binding sites, were able to originate in vivo stable complexes with Grb2. Although, Grb2-binding remains higher in both full-length isoforms, compared to the C-terminal truncated mutants, these mutants were also able to activate Ras, supporting a potential role of this C-terminal region as negative modulator of Sos1 activity. These findings document the existence of a new, functional, SH3-minimal binding site located in the specific stretch of hSos1 isoform II which may be responsible for the increased Grb2 affinity of this isoform in comparison to isoform I, and for the physiological properties differences between both isoforms. Moreover, these SH3-minimal binding sites may be sufficient to attain stable and functional hSosl-Grb2 complexes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas/metabolismo , Proteína SOS1/metabolismo , Domínios de Homologia de src/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Proteína Adaptadora GRB2 , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SOS1/genética , Proteína SOS1/fisiologia , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Domínios de Homologia de src/genéticaRESUMO
Vulval induction in Caenorhabditis elegans has helped define an evolutionarily conserved signal transduction pathway from receptor tyrosine kinases (RTKs) through the adaptor protein SEM-5 to RAS. One component present in other organisms, a guanine nucleotide exchange factor for Ras, has been missing in C.ELEGANS: To understand the regulation of this pathway it is crucial to have all positive-acting components in hand. Here we describe the identification, cloning and genetic characterization of C.ELEGANS: SOS-1, a putative guanine nucleotide exchanger for LET-60 RAS. RNA interference experiments suggest that SOS-1 participates in RAS-dependent signaling events downstream of LET-23 EGFR, EGL-15 FGFR and an unknown RTK. We demonstrate that the previously identified let-341 gene encodes SOS-1. Analyzing vulval development in a let-341 null mutant, we find an SOS-1-independent pathway involved in the activation of RAS signaling. This SOS-1-independent signaling is not inhibited by SLI-1/Cbl and is not mediated by PTP-2/SHP, raising the possibility that there could be another RasGEF.