Cell-cycle regulation of sarcomere integrity-Role for Actn2 phosphorylation

Sarcomeres are the fundamental contractile units of muscle. Despite their importance, sarcomere assembly remains poorly understood. We focused on Actn2, a protein which stabilizes the sarcomere by linking proteins to the Z-disk. During C2C12 differentiation into myoblasts, Actn2 protein levels remained constant. This finding suggested that Actn2 incorporation into the sarcomere arose from a post-translational mechanism. We hypothesized that the post-translational mechanism relied on phosphorylation. Alignment of Actn2 protein sequences from animals with three- or four-chambered hearts identified a conserved sequence, T³⁰⁸P³⁰⁹E³¹⁰K³¹¹, that matches the consensus phosphorylation motif of the cell-cycle kinase CDK1. In vitro kinase assays showed that CDK1 phosphorylates Actn2 at T308. In contrast, CDK1 was unable to phosphorylate Actn2 when T308 was mutated to alanine (T308A). Using CRISPR-Cas9 gene editing, we created Actn2-T308A and phosphomimetic Actn2-T308D variants in C2C12 cells. C2C12 cells expressing Actn2-T308A differentiated rapidly and formed robust sarcomeres. However, C2C12 cells expressing Actn2-T308D failed to form organized sarcomeres. Curiously, Actn2-T308A cells were found to have less proliferative capacity than Actn2-T308D cells. Taken together, these results identify CDK1-dependent phosphorylation of Actn2-T308 as being important for sarcomere assembly. Moreover, the data also suggest a mechanism by which cell-cycle exit promotes sarcomere assembly.