Abstract 306: A Novel PKCalpha Splicing Isoform in Signaling for Cardiac Hypertrophy

Abstract

RNA splicing is a significant post-transcriptional regulatory step to enrich the overall transcriptome complexity, yet its specific contribution to cardiac development and pathogenesis remains underexplored. Here, we performed a comprehensive deep RNA-seq analysis in murine heart to investigate the cardiac transcriptome at single exon-resolution. From the dataset, we have identified a previously un-annotated novel exon in the PKCα transcript ---PKCα-NE (Novel Exon). The novel exon encode a 16 amino acid insert adjacent to the PKCα turn motif. Interestingly, the splicing event is detected only in cardiac and skeletal muscle. Based on bioinformatics analysis, we have identified two highly conserved binding motifs for a cardiac splicing regulator_RBFox1. We further demonstrated that RBFox1 indeed functions as a positive regulator for the splicing event of this novel exon. In vitro studies showed that the NE converts PKCα from a lipid-dependent enzyme to a constitutively active kinase with high levels of autocatalytic activity and activity toward peptide substrates. The NE also increases PKCα’s Km for peptide substrate. These changes in steady state kinetic parameters suggest that PKCα-NE would exert it’s cellular actions most prominently when co-localized with target substrates, either on protein scaffolds or in subcellular signaling compartments. In fact, immune-precipitation for individual PKCα spicing variants followed by Mass Spectrometry exposed a specific interaction between PKCα-NE and elongation factor (eEF1A1). PKCα-WT and PKCα-NE both stimulated cardiac hypertrophy to a similar level based on myocyte size measurement, but PKCα-NE selectively increased eEF1A1 phosphorylation in association with elevated protein translational activity in cultured cardiomyocytes. In summary, we have identified a novel cardiac specific splicing variant of PKCα that is dynamically regulated during development and heart failure. Our finding has revealed a novel signaling pathway in cardiac hypertrophy involving a novel alternative splicing event of PKCα and its specific downstream effects on protein synthesis regulation.