RESUMEN
Cancer cells are capable of serum- and anchorage-independent growth, and focus formation on monolayers of normal cells. Previously, we showed that RACK1 inhibits c-Src kinase activity and NIH3T3 cell growth. Here, we show that RACK1 partially inhibits v-Src kinase activity, and the serum- and anchorage-independent growth of v-Src transformed cells, but has no effect on focus formation. RACK1-overexpressing v-Src cells show disassembly of podosomes, which are actin-rich structures that are distinctive to fully transformed cells. Together, our results demonstrate that RACK1 overexpression in v-Src cells partially reverses the transformed phenotype of the cells. Our results identify an endogenous inhibitor of the oncogenic Src tyrosine kinase and of cell transformation.
Asunto(s)
Proteína Oncogénica pp60(v-src)/metabolismo , Péptidos/fisiología , Actinas/metabolismo , Actinas/ultraestructura , Animales , Adhesión Celular/fisiología , Recuento de Células , División Celular/fisiología , Línea Celular Transformada , Medios de Cultivo , Proteínas del Citoesqueleto/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Ratones , Células 3T3 NIH , Proteína Oncogénica pp60(v-src)/antagonistas & inhibidores , Proteína Oncogénica pp60(v-src)/genética , Paxillin , Péptidos/genética , Péptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/metabolismo , Receptores de Cinasa C Activada , Suero , Transfección , Transformación Genética , Tirosina/metabolismoRESUMEN
RACK1 is one of a group of PKC-interacting proteins collectively called RACKs (Receptors for Activated C-Kinases). Previously, we showed that RACK1 also interacts with the Src tyrosine kinase, and is an inhibitor of Src activity and cell growth. PKC activation induces the intracellular movement and co-localization of RACK1 and Src, and the tyrosine phosphorylation of RACK1. To determine whether RACK1 is a Src substrate, we assessed phosphorylation of RACK1 by various tyrosine kinases in vitro, and by kinase-active and inactive mutants of Src in vivo. We found that RACK1 is a Src substrate. Moreover, Src activity is necessary for both the tyrosine phosphorylation of RACK1 and the binding of RACK1 to Src's SH2 domain that occur following PKC activation. To identify the tyrosine(s) on RACK1 that is phosphorylated by Src, we generated and tested a series of RACK1 mutants. We found that Src phosphorylates RACK1 on Tyr 228 and/or Tyr 246, highly-conserved tyrosines located in the sixth WD repeat that interact with Src's SH2 domain. We think that RACK1 is an important Src substrate that signals downstream of growth factor receptor tyrosine kinases and is involved in the regulation of Src function and cell growth.