Phosphorylation substrates for protein kinase C in intact pituitary cells: characterization of a receptor-mediated event using novel gonadotropin-releasing hormone analogues.
The involvement of protein kinase C in the signal transduction of gonadotropin-releasing hormone (GnRH) action was investigated with a GnRH superagonist, partial agonists, and antagonists in intact rat pituitary cells. Exposure of 32P-labeled cells to GnRH or to the superagonist [D-Nal(2)6]GnRH (200 times GnRH potency in vivo) induced the enhanced phosphorylation of 42-, 34-, 11-, and 10-kDa proteins and the dephosphorylation of a 15-kDa protein as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. This effect was blocked in a dose-dependent manner by potent GnRH antagonists. At its maximally effective concentration of 10(-9) M, [D-Nal(2)6]GnRH induced an up to 2 times more pronounced phosphorylation of endogenous substrates than GnRH at 10(-7) M. This was in accord with its ability to cause an 8-fold increase in the translocation of protein kinase C to the particulate fraction vs. 3.4-fold for GnRH. This effect correlated with potency for a series of GnRH agonists ( [D-Nal(2)6]GnRH greater than GnRH greater than [Gly2]LH-RH) and was prevented by GnRH antagonists, as assessed by a novel phorbol ester receptor binding assay and by a standard kinase assay. Downregulation of protein kinase C by prolonged incubation of the pituitary cells with high concentrations of active phorbol esters abolished protein kinase C activity and also prevented the phosphorylation induced by GnRH, or [D-Nal(2)6]GnRH. The same effect was obtained by preincubating the cells with the protein kinase C inhibitor H-7. In this study we identify for the first time physiological substrates for protein kinase C in intact pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Strulovici
B
B
Institute of Biological Sciences, Syntex Research, Palo Alto, California 94303.
Tahilramani
R
R
Nestor
J J
JJ
D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D010703:Phorbol Esters / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011955:Receptors, Drug; D013379:Substrate Specificity
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D010703:Phorbol Esters; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011506:Proteins; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D007987:Gonadotropin-Releasing Hormone; D011493:Protein Kinase C