Phosphorylation of caldesmon at sites between residues 627 and 642 attenuates inhibitory activity and contributes to a reduction in Ca2+-calmodulin affinity.
Caldesmon 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 Ca(2+)-calmodulin. We investigated the effects of phosphorylation by p(21)-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 Ca(2+)-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 Ca(2+)-calmodulin binding further and reduced the inhibitory activity of CaD22 in the absence of Ca(2+)-calmodulin. Phosphorylation of these sites of the alanine mutant of CaD22 had no effect on Ca(2+)-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.
Hamden
Svetlana S
SS
Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina, USA.
Schroeter
Mechthild M
MM
Chalovich
Joseph M
JM
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002645:Chickens; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000235:genetics; D011485:Protein Binding; D011817:Rabbits
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D000251:Adenosine Triphosphatases; D002118:Calcium