Abstract 1073: A systematic approach to identify the Ras-dependent C-RAF(Ser338)-phosphorylating kinase

Phosphorylation of c-RAF at Ser338 is an absolute requirement for c-RAF activation and Ras-mediated signaling; however, definitive identification of the c-RAF(Ser338)-phosphorylating kinase has remained elusive. As a first step towards this end, we screened a collection of ∼600 expressible kinase open reading frames to determine which are sufficient to induce phosphorylation of c-RAF on Ser338. In total, 12 kinases induced c-RAF(Ser338) phosphorylation, including 9 Ser/Thr kinases, 1 kinase with unknown site specificity, 1 adapter factor and 1 scaffolding protein. Notably, two proteins previously shown to regulate c-RAF(Ser338) phosphorylation (PAK3 and KSR) also scored in this assay, highlighting the utility of this approach to uncover plausible effectors. We have initiated follow-up studies, including recombinant protein-based kinase assays and loss of function experiments, to characterize the kinase(s) responsible for Ras-mediated c-RAF(Ser338) phosphorylation in vivo. Given the inherent difficulty of targeting Ras-dependent cancers, a more detailed understanding of c-RAF phosphorylation and Ras-dependent activation may inform new therapeutic inroads to targeting these lesions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1073. doi:10.1158/1538-7445.AM2011-1073