Phosphorylation of Anchoring Protein by Calmodulin Protein Kinase Associated to the Sarcoplasmic Reticulum of Rabbit Fast-Twitch Muscle

Regulatory phosphorylation of phospholamban and of SR Ca2+-ATPase SERCA2a isoform by endogenous CaM-K II in slow-twitch skeletal and cardiac sarcoplasmic reticulum (SR) is well documented, but much less is known of the exact functional role of CaM K II in fast-twitch muscle SR. Recently, it was shown that RNA splicing of brain-specific α CaM K II, gives rise to a truncated protein (αKAP), consisting mainly of the association domain, serving to anchor CaM K II to SR membrane in rat skeletal muscle [Bayer, K.-U., et al. (1998) EMBO J. 19, 5598–5605]. In the present study, we searched for the presence of αKAP in sucrose-density purified SR membrane fractions from representative fast-twitch and slow-twitch limb muscles, both of the rabbit and the rat, using immunoblot techniques and antibody directed against the association domain of α CaM K II. Putative αKAP was immunodetected as a 23-kDa electrophoretic component on SDS–PAGE of the isolated SR from fast-twitch but not from slow-twitch muscle, and was further identified as a specific substrate of endogenous CaM K II, in the rabbit. Immunodetected, 32P-labeled, non-calmodulin binding protein, behaved as a single 23-kDa protein species under several electrophoretic conditions. The 23-kDa protein, with defined properties, was isolated as a complex with 60-kDa δ CaM K II isoform, by sucrose-density sedimentation analysis. Moreover, we show here that putative αKAP, in spite of its inability to bind CaM in ligand blot overlay, co-eluted with δ CaM K II from CaM-affinity columns. That raises the question of whether CaM K II-mediated phosphorylation of αKAP and triadin together might be involved in a molecular signaling pathway important for SR Ca2+-release in fast-twitch muscle SR.