Phosphorylation of Caldesmon at Sites between Residues 627 and 642 Attenuates Inhibitory Activity and Contributes to a Reduction in Ca2+-Calmodulin Affinity

AbstractCaldesmon is an actin- and myosin-binding protein found in smooth muscle that inhibits actin activation of myosin ATPase activity. The activity of caldesmon is controlled by phosphorylation and by binding to Ca2+-calmodulin. We investigated the effects of phosphorylation by p21-activated kinase 3 (PAK) and calmodulin on the 22 kDa C-terminal fragment of caldesmon (CaD22). We substituted the major PAK sites, Ser-672 and Ser-702, with either alanine or aspartic acid to mimic nonphosphorylated and constitutively phosphorylated states of caldesmon, respectively. The aspartic acid mutation of CaD22 weakened Ca2+-calmodulin binding but had no effect on inhibition of ATPase activity. Phosphorylation of the aspartic acid mutant with PAK resulted in the slow phosphorylation of Thr-627, Ser-631, Ser-635, and Ser-642. Phosphorylation at these sites weakened Ca2+-calmodulin binding further and reduced the inhibitory activity of CaD22 in the absence of Ca2+-calmodulin. Phosphorylation of these sites of the alanine mutant of CaD22 had no effect on Ca2+-calmodulin binding but did reduce inhibition of ATPase activity. Thus, the region between residues 627 and 642 may contribute to the overall regulation of caldesmon's activity