Phosphorylation of calmodulin fragments by protein kinase CK2. Mechanistic aspects and structural consequences.

Calmodulin is phosphorylated in vivo and in vitro by protein kinase CK2 in a manner that is unique among CK2 substrates for being inhibited by the regulatory beta-subunit of the kinase and dramatically enhanced by polybasic peptides. Using synthetic fragments of calmodulin variably encompassing the CK2 phosphorylation sites here we show that individual phosphorylation of Thr79, Ser81, Ser101, and Thr117 is critically influenced by the size and composition of the peptides and that the C-terminal domain of calmodulin is implicated both in down-regulation of calmodulin phosphorylation by the beta-subunit and in its abnormal responsiveness to polylysine. A far-Western blot analysis discloses polylysine-dependent interaction between calmodulin and the N-terminal domain of the beta-subunit. We also show that phosphorylation of Ser81 hampers subsequent phosphorylation of Thr79 and by itself promotes the unfolding of the central helix, whose flexibility is instrumental to the interaction with calmodulin-dependent enzymes. Collectively taken, our data are consistent with a multifaceted regulation of calmodulin phosphorylation through the concerted action of distinct CaM domains, the catalytic and regulatory subunits of CK2, and polycationic effectors mimicking in vivo the effect of polylysine.