Phosphorylation of a chromaffin granule-binding protein in stimulated chromaffin cells.
A procedure was devised to determine whether in the stimulated chromaffin cell phosphate is incorporated into specific proteins ("chromobindins") that bind to chromaffin granule membranes in a Ca2+-dependent manner. Cells were preincubated with 32P-labeled orthophosphate, then challenged with secretory stimuli. A postmicrosomal supernatant fraction was prepared from the cells and incubated with unlabeled chromaffin granule membranes in the presence of 5 mM Ca2+. Proteins that bound to the membranes were isolated by centrifugation and examined for 32P content by electrophoresis and autoradiography. Stimulation by carbamylcholine, nicotine, 56 mM K+, or 2 mM Ba2+ led to the incorporation of 32P into a 37-kDa protein that had previously been characterized as a substrate for protein kinase C in vitro (chromobindin 9, or CB9; Summers, T. A., and Creutz, C. E. (1985) J. Biol. Chem. 260, 2437-2443). Incorporation of 32P into this protein was dependent on extracellular Ca2+ and followed a time course that paralleled secretion of catecholamines, returning to base-line levels after 30 min, when secretion terminated. 32P was also incorporated into a 58-kDa protein that may be tyrosine hydroxylase and into an unidentified 28-kDa protein in response to cell stimulation, but neither of these proteins bound to granule membranes in a Ca2+-dependent manner. Treatment of cells with phorbol 12,13-dibutyrate, an activator of protein kinase C, led to 32P incorporation into the 37-kDa protein that was only 30% of the level obtained with nicotinic stimulation, suggesting that additional kinases may be involved in phosphorylating this protein in the stimulated cell.
M L
ML
Michener
W B
WB
Dawson
C E
CE
Creutz
D000818:Animals; D017302:Annexins; D002217:Carbachol / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D002838:Chromaffin System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007093:Imidazoles / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D009538:Nicotine / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation
D017302:Annexins; D002352:Carrier Proteins; D007093:Imidazoles; D008565:Membrane Proteins; D010718:Phosphatidylserines; D010750:Phosphoproteins; C031938:calmidazolium; D009538:Nicotine; D002217:Carbachol