Phosphorylation of dystrophin and alpha-syntrophin by Ca2+-calmodulin dependent protein kinase II

A Ca2+-calmodulin dependent protein kinase activity (DGC-PK) was previously shown to associate with skeletal muscle dystrophin glycoprotein complex (D GC) preparations, and phosphorylate dystrophin and a protein with the same electrophoretic mobility as alpha-syntrophin (R. Madhavan, H.W. Jarrett, Bi ochemistry 33 (1994) 5797-5804). Here, we show that DGC-PK and Ca2+-calmodu lin dependent protein kinase II (CaM kinase II) phosphorylate a common site (RSDS3616) within the dystrophin C terminal domain that fits the consensus CaM kinase II phosphorylation motif (R/KXXS/T). Furthermore, both kinase a ctivities phosphorylate exactly the same three fusion proteins (dystrophin fusions DysS7 and DysS9, and the syntrophin fusion) out of a panel of eight fusion proteins (representing nearly 100% of syntrophin and 80% of dystrop hin protein sequences), demonstrating that DGC-PK and CaM kinase II have th e same substrate specificity. Complementing these results, anti-CaM kinase II antibodies specifically stained purified DGC immobilized on nitrocellulo se membranes. Renaturation of electrophoretically resolved DGC proteins rev ealed a single protein kinase band (M-r approximate to 60 000) that, like C aM kinase II, underwent Ca2+-calmodulin dependent autophosphorylation. Base d on these observations, we conclude DGC-PK represents a dystrophin-/syntro phin-phosphorylating skeletal muscle isoform of CaM kinase II. We also show that phosphorylation of the dystrophin C terminal domain sequences inhibit s their syntrophin binding in vitro, suggesting a regulatory role for phosp horylation. (C) 1999 Elsevier Science B.V. All rights reserved.