Abstract 1245: Polyribosome association of tyrosine phosphorylated RACK1 with activated Src regulates translation of specific proteins mediating breast cancer growth and metastasis

Dysregulation of translational control, characterized by aberrant expression of proteins critical for growth and metastatic potential, is emerging as an essential aspect of tumorigenesis. Receptor for activated protein kinase C-1 (RACK1) is a scaffold protein known to coordinate the cellular localization of numerous signaling proteins including PKC and Src, and has recently been identified as an essential ribosome component binding to the head region of the 40S subunit. RACK1 was identified in a mass spectrometry screen of polyribosome proteins from human breast cancer cells demonstrating altered mobility on 2D gel electrophoresis following growth-arresting doses of a histone deacetylase (HDAC) inhibitor which suggested that post-translational modifications of ribosome associated RACK1 might selectively influence protein synthesis and cancer cell growth. Polyribosome profiling of MDA-231 and SKBR3 breast cancer cells revealed that while the entire complement of lysate soluble RACK1 was ribosome associated in both cell lines, only RACK1 from the MDA-231 cells appeared significantly tyrosine phosphorylated. Furthermore, tyrosine phosphorylated Src (pTyr-416), while 10 fold more abundant in the SKBR3 untranslated fractions relative to those from MDA-231, was associated with the polyribosome factions of MDA-231 but not those of SKBR3, consistent with previous observations linking activated Src with tyrosine phosphorylated RACK1. Lentiviral delivery of shRNA was used to investigate the functional consequences of selective RACK1 knockdown. In MDA-231 cells, efficient RACK1 knockdown produced complete growth arrest and loss of motility without induction of apoptosis or cell senescence; however, this growth arrested phenotype is associated with near complete loss of cyclin D1 and TGFbetaR1 expression without change in the expression of housekeeping proteins beta-actin and GAPDH. Taken together, these results implicate tyrosine phosphorylated RACK1 in association with polysomal pTyr-Src as translational activators of specific proteins mediating breast cancer proliferation and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1245.