Ankrd2/ARPP is a Novel Akt2 Specific Substrate and Regulates Myogenic Differentiation Upon Cellular Exposure to H2O2.

Activation of Akt-mediated signaling pathways is crucial for survival, differentiation and regeneration of muscle cells. A proteomic–based search for novel substrates of Akt was therefore undertaken in C2C12 murine muscle cells exploiting protein characterization database in combination with an anti phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Serine 99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells where mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H2O2 triggers this phosphorylation. Moreover, the forced expression of a phosphorylation defective mutant form of Ankrd2 in C2C12 myoblasts, promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Serine 99 in the negative regulation of myogenesis in response to stress conditions.