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

Abstract

We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by PKBα (protein kinase Bα), but not by SGK1 (serum- and glucocorticoid-induced kinase 1), and identified it as the cytoskeletal protein FLNc (filamin C). PKBα phosphorylated FLNc at Ser²²¹³ in vitro, which lies in an insert not present in the FLNa and FLNb isoforms. Ser²²¹³ 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 rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] had no effect. Insulin also induced the phosphorylation of FLNc at Ser²²¹³ 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.