Identification of filamin C as a new, physiological substrate of PKBα using KESTREL

We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by PKBalpha (protein kinase Balpha), but not by SGK1 (serum- and glucocorticoid-induced kinase 1), and identified it as the cytoskeletal protein FLNc (filamin C). PKBalpha phosphorylated FLNc at Ser(2213) in vitro, which ties in an insert not present in the FLNa and FLNb isoforms. Ser(2213) became phosphorylated when C2C12 myoblasts were stimulated with insulin or epidermal growth factor, and phosphorylation was prevented by low concentrations of wortmannin, at which it is a relatively specific inhibitor of phosphoinositide 3-kinase. PD 184352 [an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade] and/or raparmycin [an inhibitor of mTOR (mammalian target of rapamycin)] had no effect. Insulin also induced the phosphorylation of FLNc at Ser(2213) in cardiac muscle in vivo, but not in cardiac muscle that does not express PDK1 (3-phosphoinositide-dependent kinase 1), the upstream activator of PKB. These results identify the muscle-specific isoform FLNc as a new physiological substrate for PKB.