Phosphorylation of liver plasma membrane-bound calmodulin.
In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [gamma-32P]ATP as phosphate donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the Ca2+-calmodulin-dependent enzyme, (3':5'AMP)-phosphodiesterase, by the EGTA extract, and (iii) electrophoretic comigration of EGTA-extracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32P-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis.
Ghosh
S
S
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Church
J G
JG
Roufogalis
B D
BD
Villalobo
A
A
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004533:Egtazic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D007158:Immunologic Techniques; D008099:Liver / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats
D002147:Calmodulin; D008565:Membrane Proteins; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002118:Calcium