RESUMO
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
Two signaling pathways known to be essential for progression from immature to mature B cells are BAFF receptor (BAFF-R) and the B cell receptor (BCR). Here, we first show that phospholipase C (PLC)-gamma2 is required for a BAFF-R-mediated survival signal. Then, we have examined the question of whether the reduced number of mature B cells in PLC-gamma2-/- mice is caused by a defect in either BCR or BAFF-R signaling. We find that a PLC-gamma2 SH2 mutant, which inhibits coupling between BCR and PLC-gamma2, fails to restore B cell maturation, despite supporting BAFF-dependent survival. Therefore, our data suggest that the BAFF-R-mediated survival signal, provided by PLC-gamma2, is not sufficient to promote B cell maturation, and that, in addition, activation of PLC-gamma2 by BCR is required for B cell development.
Assuntos
Linfócitos B/fisiologia , Proteínas de Membrana , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Receptor do Fator Ativador de Células B , Linfócitos B/citologia , Células da Medula Óssea/fisiologia , Sobrevivência Celular , Genes bcl-2 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Fosfolipase C gama , Transdução de Sinais/fisiologia , Baço/citologia , Fosfolipases Tipo C/genéticaRESUMO
Despite the importance of the Vav family proteins for B cell receptor (BCR) signaling, their activation mechanisms remain poorly understood. We demonstrate here that adaptor molecules Grb2 and BLNK, in addition to Vav, are required for efficient Rac1 activation in response to BCR stimulation. Loss of either Grb2 or BLNK results in decreased translocation of Vav3 to membrane rafts. By expression of Vav3 as a raft-targeted construct, the defective Rac1 activation in Grb2- or BLNK-deficient B cells is restored. Hence, our findings suggest that Grb2 and BLNK cooperate to localize Vav into membrane rafts, thereby contributing to optimal activation of Vav in B cells.