The Proto-oncogene PKCι Regulates the Alternative Splicing of Bcl-x Pre-mRNA

Abstract

Two splice variants derived from the BCL-x gene via alternative 5′ splice site selection (5′SS) are pro-apoptotic Bcl-x(s) and anti-apoptotic Bcl-x(L). Previously, our laboratory demonstrated that apoptotic signaling pathways regulated the alternative 5′SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive pro-survival signaling pathways that regulate the 5′SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by utilizing 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 phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (50 μM) or the pan-PKC inhibitor GÖ6983 (25 μM) decreased the Bcl-x(L)/Bcl-x(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/Bcl-x(s) mRNA ratio. Additional studies demonstrated that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/Bcl-x(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5′SS 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. Lastly, forced expression of Bcl-x(L) “rescued” the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.

Caspase-9b Interacts Directly with cIAP1 to Drive Agonist-Independent Activation of NF-κB and Lung Tumorigenesis

“…Quantitative RT-PCR (RT-qPCR) were performed as described (19,20,23,24) utilizing primers for the genes listed in Table S1. The relative mRNA expression was normalized to 18S except for human tissues where relative mRNA expression was normalized to ESD.…”

Protein Kinase C (PKC) Isozymes and Cancer

“…The survival mechanisms of SCLCs and NSCLCs through PKC involve upregulation of the antiapoptotic protein Bcl-XL [310] and S-phase kinase-associated protein 2-mediated anoikis resistance via the PI3K/Akt pathway [308]. PKC also regulates transformed growth and invasion of NSCLC cells mainly through the Rac1/PAK/MEK/ERK signaling pathway [307,311,312].…”

Melanoma Differentiation-associated Gene 7/IL-24 Exerts Cytotoxic Effects by Altering the Alternative Splicing of Bcl-x Pre-mRNA via the SRC/PKCδ Signaling Axis

“…This was a novel finding as this is in contrast to previous signaling pathways reported to regulate Bcl-x RNA splicing in cancer such as the PI3K/PKC pathway or PKC␣ signaling in non-cancerous cells (see for example Refs. 24,25,71,72). One may surmise that MDA-7/IL-24 logically acts to drive terminal differentiation pathways, explaining the SRC activation paradigm as well as PKC␦, which are implicated in a number of differentiation processes.…”