In vitro phosphorylation of bovine cardiac muscle high molecular weight calmodulin binding protein by cyclic AMP-dependent protein kinase and dephosphorylation by calmodulin-dependent phosphatase

摘要

High molecular weight calmodulin binding protein (HMWCaMBP) is one of the major proteins expressed in bovine cardiac muscle [9]. In this study, we report the phosphorylation and dephosphorylation of HMWCaMBP in vitro with a view to understand the function of this protein. The HMWCaMBP was phosphorylated by cAMP-dependent protein kinase with the incorporation of 2.30 mol of phosphate/mol of protein in the presence of EGTA. When phosphorylation was carried out in the presence of Ca2+/calmodulin (CaM), the incorporation of phosphate was reduced to 1.40 mol of phosphate/mol of protein. The decrease in the stoichometry of phosphorylation by Ca2+/CaM appears to be substrate directed i.e. due to the interaction of Ca2+/CaM with HMWCaMBP. The phosphorylated HMWCaMBP was unable to compete for free CaM in a CaM-dependent cyclic nucleotide phosphodiesterase (CaMPDE) assay. These results suggest that the phosphorylation sites may reside in or in proximity to the CaM-binding domain on HMWCaMBP since phosphorylated HMWCaMBP did not inhibit CaMPDE activity. HMWCaMBP was dephosphorylated by CaM-dependent phosphatase, calcineurin.