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 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 duanyu1531 at the nuclear level during myogenesis and to find new duanyu1531 isozyme-specific phosphosubstrates, we performed a proteomics analysis of immunoprecipitated nuclear samples from mouse myoblast C2C12 cells following insulin administration. Using a 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 substrate antibody were identified; among these, particular interest was given to eukaryotic elongation factor 1α (eEF1A). Nuclear eEF1A was focalized in the nucleoli, and its expression was observed to increase following insulin treatment. Of the isoforms, only 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 duanyu1531βI could phosphorylate Ser⁵³ of the eEF1A2 isoform and that the association between eEF1A2 and duanyu1531βI was dependent on the phosphorylation status of eEF1A2.

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