In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase.
Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.
Bookbinder
L H
LH
Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla 92093.
Moy
G W
GW
Vacquier
V D
VD
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003488:Cyanogen Bromide / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D012617:Sea Urchins
D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D011494:Protein Kinases; D000262:Adenylyl Cyclases; D003488:Cyanogen Bromide; D002118:Calcium