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.
High molecular weight calmodulin binding protein (HMWCaMBP) is one of the major proteins expressed in bovine cardiac muscle. 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.
Kakkar
R
R
Department of Pathology and Saskatoon Cancer Centre, College of Medicine, University of Saskatchewan, Canada.
Taketa
S
S
Raju
R V
RV
Proudlove
S
S
Colquhoun
P
P
Grymaloski
K
K
Sharma
R K
RK
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D019703:Calcineurin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004789:Enzyme Activation; D009206:Myocardium / Q000201:enzymology* / Q000378:metabolism*; D010766:Phosphorylation
D002148:Calmodulin-Binding Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D019703:Calcineurin; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1