The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain.
The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32P from [gamma-32P]ATP into calmodulin fragment 1-106. Incorporation was exclusively into serine 101. With fragment 78-148, the extent of phosphorylation was somewhat less and 32P appeared mainly in threonine residues. Fragment 1-90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca2+-binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg2+ (60-70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble calmodulin.
Heppel
L A
LA
Laboratory of Biochemistry, National Cancer Institute, Bethesda, MD 20892.
Newton
D L
DL
Klee
C B
CB
Draetta
G F
GF
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D002147:Calmodulin; D002413:Cations, Divalent; D010446:Peptide Fragments; D010710:Phosphates; D000242:Cyclic AMP; D011494:Protein Kinases; D005459:Fluorides; D002118:Calcium