Follicle-Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-lnduced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells.
FSH stimulates in ovarian granulosa cells diverse, differentiation-dependent responses that implicate activation of specific cellular signaling cascades. In these studies three kinases were investigated to determine their relationship to FSH, cAMP, and A kinase signaling: protein kinase B (PKB/Akt), serum and glucocorticoid-induced kinase (Sgk), and p38 mitogen-activated protein kinase (p38MAPK). The phosphorylation (activation) of these kinases was analyzed by using selective agonists/inhibitors: forskolin/H89 for cAMP-dependent protein kinase (A kinase), insulin-like growth factor I (IGF-I)/LY294002 and wortmannin for phosphatidylinositol-dependent kinase (PI3-K), and phorbol myristate (PMA)/GF109203X for diacylglycerol and Ca++-dependent kinases (C kinases). An inhibitor (PD98059) of MEK1, which regulates extracellular regulated kinases (ERKs), and SB203580, which inhibits p38MAPK, were also used. In addition, we analyzed the expression of the recently described, cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFI and GEFII) that impact Ras-related GTPases and Raf kinases, known regulators of various protein kinase cascades. We provide evidence that FSH, forskolin, and 8-bromo-cAMP stimulate phosphorylation of PKB by mechanisms involving PI3-K (LY294002/wortmannin sensitive) not A kinase (H89 insensitive), a pattern of response mimicking that of IGF-I. In contrast, FSH induction and phosphorylation of Sgk protein requires A kinase (H89 sensitive) but also involves PI3-K (LY294002 sensitive) as well as p38MAPK (SB203580 sensitive) pathways. PMA (C kinase) abolished FSH-mediated (but not IGF-I-mediated) phosphorylation of PKB at a step(s) upstream of PI3-K and independent of A kinase. Lastly, FSH-mediated phosphorylation of p38MAPK is negatively affected by A kinase and PI3-K, suggesting that it may be downstream of specific members of the cAMP-GEF/Rap/Raf pathway. We propose that cAMP activation of A kinase is obligatory for transcription of Sgk in granulosa cells whereas cAMP (IGF-I-like)-mediated phosphorylation (activation) of PKB and Sgk (via PI3-K), as well as p38MAPK, involves other cellular events. These results provide new and exciting evidence that cAMP acts in granulosa cells by A kinase-dependent and -independent mechanisms, each of which controls specific kinase cascades.
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
I J
IJ
Gonzalez-Robayna
A E
AE
Falender
S
S
Ochsner
G L
GL
Firestone
J S
JS
Richards
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism* / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D017874:Immediate-Early Proteins; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009687:Nuclear Proteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D015800:Protozoan Proteins / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013739:Testosterone / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases; D000099184:Serum-Glucocorticoid Regulated Kinases
D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005640:Follicle Stimulating Hormone; D020662:Guanine Nucleotide Exchange Factors; D017874:Immediate-Early Proteins; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D009687:Nuclear Proteins; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D015800:Protozoan Proteins; D013739:Testosterone; D013755:Tetradecanoylphorbol Acetate; D048051:p38 Mitogen-Activated Protein Kinases; D000099184:Serum-Glucocorticoid Regulated Kinases; C116122:RAPGEF3 protein, human; C065352:rhoptry associated protein, Plasmodium; C073168:rhoptry-associated antigen-2, Plasmodium; C494920:Akt1 protein, rat; C482125:MAP2K1 protein, human