eEF1A Phosphorylation in the Nucleus of Insulin-stimulated C2C12 Myoblasts SER53 IS A NOVEL SUBSTRATE FOR PROTEIN KINASE C βI

Recent data indicate that some PKC isoforms are translocated to the nucleus, in response to certain stimuli, where they play an important role in nuclear signaling events. To identify novel interacting proteins of conventional PKC (cPKC) at the nuclear level during myogenesis and to find new PKC isozyme-specific phosphosubstrates, we performed a proteomics analysis of immunoprecipitated nuclear samples from mouse myoblast C2C12 cells following insulin administration. Using a phospho(Ser)-PKC substrate antibody, specific interacting proteins were identified by LC-MS/MS spectrometry. A total of 16 proteins with the exact and complete motif recognized by the phospho-cPKC substrate antibody were identified; among these, particular interest was given to eukaryotic elongation factor 1 alpha (eEF1A). Nuclear eEF1A was focalized in the nucleoli, and its expression was observed to increase following insulin treatment. Of the cPKC isoforms, only PKC beta I was demonstrated to be expressed in the nucleus of C2C12 myocytes and to co-immunoprecipitate with eEF1A. In-depth analysis using site-directed mutagenesis revealed that PKC beta I could phosphorylate Ser(53) of the eEF1A2 isoform and that the association between eEF1A2 and PKC beta I was dependent on the phosphorylation status of eEF1A2.