RESUMEN
The Epstein-Barr virus latency-associated membrane protein LMP2A has been shown to activate the survival kinase Akt in epithelial and B cells in a phosphoinositide 3-kinase-dependent fashion. In this study, we demonstrate that the signalling scaffold Shb associates through SH2 and PTB domain interactions with phosphorylated tyrosine motifs in the LMP2A N-terminal tail. Additionally, we show that mutation of tyrosines in these motifs as well as shRNA-mediated downregulation of Shb leads to a loss of constitutive Akt-activation in LMP2A-expressing cells. Furthermore, utilization by Shb of the LMP2A ITAM motif regulates stability of the Syk tyrosine kinase in LMP2A-expressing cells. Our data set the precedent for viral utilization of the Shb signalling scaffold and implicate Shb as a regulator of LMP2A-dependent Akt activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfocitos B/virología , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Proteínas de la Matriz Viral/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Sitios de Unión , Línea Celular , Línea Celular Transformada , Herpesvirus Humano 4/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Represoras/metabolismo , Quinasa Syk , Tirosina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de la Matriz Viral/químicaRESUMEN
Recent work has implicated the importance of adapter proteins in signal transduction. To identify homologues of the previously identified adapter protein Shb, database searches were performed. A Shb-like protein was found which we have named Shf. Shf contains an SH2 domain and four putative tyrosine phosphorylation sites and is mainly expressed in skeletal muscle, brain, liver, prostate, testis, ovary, small intestine, and colon. The SH2 domain of Shf bound to the PDGF-alpha-receptor at tyrosine-720, but not to the PDGF-beta-receptor in PAE cells. Pervanadate induced tyrosine phosphorylation of Shf in NIH3T3 fibroblasts overexpressing this protein, whereas PDGF-AA alone had no detectable effect. NIH3T3 cells overexpressing Shf displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. Our findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis/fisiología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/fisiología , Células 3T3 , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/fisiología , Clonación Molecular , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfotirosina/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Vanadatos/farmacología , Dominios Homologos srcRESUMEN
Stimulation of the T cell antigen receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins. We have recently investigated the role of the adaptor protein Shb in the early events of T cell signaling and observed that Shb associates with Grb2, linker for activation of T cells (LAT) and the TCR zeta-chain in Jurkat cells. We now report that Shb also associates with phospholipase C-gamma1 (PLC-gamma1) in these cells. Overexpression of Src homology 2 domain defective Shb caused diminished phosphorylation of LAT and consequently the activation of mitogen-activated protein kinases was decreased upon TCR stimulation. In addition, the Shb mutant also blocked phosphorylation of PLC-gamma1 and the increase in cytoplasmic Ca(2+) following TCR stimulation. Nuclear factor for activation of T cells is a major target for Ras and calcium signaling pathways in T cells following TCR stimulation, and the overexpression of the mutant Shb prevented TCR-dependent activation of the nuclear factor for activation of T cells. Consequently, endogenous interleukin-2 production was decreased under these conditions. The results indicate a role for Shb as a link between the TCR and downstream signaling events involving LAT and PLC-gamma1 and resulting in the activation of transcription of the interleukin-2 gene.