RESUMEN
The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis. Cbp-deficient cells are more susceptible to v-Src transformation than their parental cells. Upon phosphorylation, Cbp specifically binds to activated c-Src and sequesters it in lipid rafts, resulting in an efficient suppression of c-Src function independent of Csk. In some human cancer cells and tumors, Cbp is downregulated and the introduction of Cbp significantly suppresses tumorigenesis. These findings indicate a potential role for Cbp as a suppressor of c-Src-mediated tumor progression.
Asunto(s)
Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Proteína Tirosina Quinasa CSK , Fraccionamiento Celular , Línea Celular Tumoral , Transformación Celular Neoplásica , Células Cultivadas , Fibroblastos/citología , Fibroblastos/fisiología , Técnicas de Transferencia de Gen , Humanos , Microdominios de Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Neoplasias/metabolismo , Fosfoproteínas/genética , Proteínas Tirosina Quinasas/genética , Familia-src QuinasasRESUMEN
To elucidate the regulatory mechanism of cell transformation induced by c-Src tyrosine kinase, we performed a proteomic analysis of tyrosine phosphorylated proteins that interact with c-Src and/or its negative regulator Csk. The c-Src interacting proteins were affinity-purified from Src transformed cells using the Src SH2 domain as a ligand. LC-MS/MS analysis of the purified proteins identified general Src substrates, such as focal adhesion kinase and paxillin, and ZO-1/2 as a transformation-dependent Src target. The Csk binding proteins were analyzed by a tandem affinity purification method. In addition to the previously identified Csk binding proteins, including Cbp/PAG, paxillin, and caveolin-1, we found that ZO-1/2 could also serve as a major Csk binding protein. ZO-2 was phosphorylated concurrently with Src transformation and specifically bound to Csk in a Csk SH2 dependent manner. These results suggest novel roles for ZO proteins as Src/Csk scaffolds potentially involved in the regulation of Src transformation.