Evidence that cyclic adenosine 3',5'-monophosphate-dependent protein kinase activation causes pig ovarian granulosa cell differentiation, including increases in two type II subclasses of this kinase.
Agents that elevated intracellular cyclic adenosine 3',5'-monophosphate (cAMP) caused a 3- to 10-fold increase in the luteinizing hormone (LH) receptor level and in progesterone biosynthesis in primary cultures of pig ovarian granulosa cells. Associated with these effects was a 2- to 4-fold increase in the total activity of the catalytic subunit of cAMP-dependent protein kinase in the tissue. From quantitation by [3H]cAMP binding and changes in the specific labeling with the photoaffinity analog [32P]-8-azido-cAMP, these agents were found to cause a concomitant 5- to 15-fold increase in two isoforms of the type II R-subunit (Mr = 54,000 and 56,000) of the protein kinase. Since the two intrasubunit cAMP binding sites of the protein kinase have been found to be positively cooperative, the addition of a combination of an analog selective for site 1 and an analog selective for site 2 causes synergistic increases in protein kinase activation in vitro and synergistic increases in intact cell responses if mediated by the cAMP-dependent protein kinase. In the present study, the addition of such a combination of site 1- and site 2-selective analogs to granulosa cells caused a synergistic increase in LH receptor induction and progesterone production. For both responses, synergism did not occur when two analogs selective for the same site were combined. The results indicated that these responses are mediated by either of the two major isozyme types of cAMP-dependent protein kinase.
Beebe
S J
SJ
Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Segaloff
D L
DL
Burks
D
D
Beasley-Leach
A
A
Limbird
L E
LE
Corbin
J D
JD
D000818:Animals; D001345:Autoradiography; D002454:Cell Differentiation; D000242:Cyclic AMP / Q000378:metabolism; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006107:Granulosa Cells / Q000166:cytology / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism*; D010766:Phosphorylation; D011374:Progesterone / Q000096:biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D011974:Receptors, LH / Q000032:analysis; D013552:Swine
D007527:Isoenzymes; D011974:Receptors, LH; D011374:Progesterone; D000242:Cyclic AMP; D011494:Protein Kinases