Signaling and Regulation TheProto-oncogene PKCiRegulates the Alternative Splicing of Bcl-x Pre-mRNA

Two splice variants derived from the Bcl-x gene via alternative 50 splice site selection (50SS) are proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L). Previously, our laboratory showed that apoptotic signaling pathways regulated the alternative 50SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive prosurvival signaling pathways that regulate the 50SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broadbased approach by using a number of small-molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non–small cell lung cancer (NSCLC) cell lines with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (50 mmol/L) or the pan-protein kinase C (PKC) inhibitor G€o6983 (25 mmol/L) decreased the Bcl-x(L)/(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCi and PKCz, had no effect on the Bcl-x(L)/(s) mRNA ratio. Additional studies showed that downregulation of the proto-oncogene, PKCi, in contrast to PKCz, also resulted in a decrease in the Bcl-x(L)/(s) mRNA ratio. Furthermore, downregulation of PKCi correlatedwith a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 50SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Finally, forced expression of Bcl-x(L) "rescued" the loss of cell survival induced by PKCi siRNA. In summary, the PI3K/PKCi regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.Mol Cancer Res; 10(5); 660–9.