Phosphorylation of a chromaffin granule-binding protein by protein kinase C.
Protein kinase C was detected in a group of Ca2+-dependent chromaffin granule membrane-binding proteins (chromobindins) on the basis of Ca2+-, phosphatidylserine-, 1,2-diolein-, and phorbol myristate acetate-stimulated histone kinase activity. When the chromobindins were incubated with [gamma-32P]ATP, Ca2+, and phosphatidylserine, 32P was incorporated predominantly into a protein of mass 37 +/- 1 kilodaltons (chromobindin 9, or CB9). Phosphorylation of this protein was also stimulated by diolein and phorbol myristate acetate, indicating that it is a substrate for the protein kinase C activity present in the chromobindins. Maximum phosphate incorporation into CB9 in the presence of 1 mM Ca2+, 75 micrograms/ml of phosphatidylserine, 2.5 micrograms/ml of diolein, and 12.5 micrograms/ml of dithiothreitol was 0.53 mol/mol of CB9 in 5 min. Eight 32P-labeled phosphopeptides were resolved in two-dimensional electrophoretic maps of trypsin digests of CB9. Phosphoamino acid analysis revealed that phosphorylation was exclusively on serine (94%) and threonine (6%) residues. Incubation of the chromobindins with chromaffin granule membranes in the presence of [gamma-32P]ATP resulted in the incorporation of 32P into eight additional proteins besides CB9 that could be separated from the membranes by centrifugation in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We suggest that phosphorylation of CB9 or these additional eight proteins may regulate events underlying exocytosis in the chromaffin cell.
Summers
T A
TA
Creutz
C E
CE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D017302:Annexins; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D004075:Diglycerides / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D010718:Phosphatidylserines / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C; D011494:Protein Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism
D017302:Annexins; D002352:Carrier Proteins; D004075:Diglycerides; D008565:Membrane Proteins; D010718:Phosphatidylserines; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; C013965:diolein