Mechanism of mitogen-activated protein kinase activation by gonadotropin-releasing hormone in the pituitary of alphaT3-1 cell line: differential roles of calcium and protein kinase C.
The mechanism of mitogen-activated protein kinase (MAPK, ERK) stimulation by the GnRH analog [D-Trp6]GnRH (GnRH-a) was investigated in the gonadotroph-derived alphaT3-1 cell line. GnRH-a as well as the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated a sustained response of MAPK activity, whereas epidermal growth factor (EGF) stimulated a transient response. MAPK kinase (MEK) is also activated by GnRH-a, but in a transient manner. GnRH-a and TPA apparently activated mainly the MAPK isoform ERK1, as revealed by Mono-Q fast protein liquid chromatography followed by Western blotting as well as by gel kinase assay. GnRH-a and TPA stimulated the tyrosine phosphorylation of several proteins, and this effect as well as the stimulation of MAPK activity were inhibited by the PKC inhibitor GF 109203X. Similarly, down-regulation of TPA-sensitive PKC subspecies nearly abolished the effect of GnRH-a and TPA on MAPK activity. Furthermore, the protein tyrosine kinase (PTK) inhibitor genistein inhibited protein tyrosine phosphorylation and reduced GnRH-a-stimulated MAPK activity by 50%, suggesting the participation of genistein-sensitive and insensitive pathways in GnRH-a action. Although Ca2+ ionophores have only a marginal stimulatory effect, the removal of Ca2+ markedly reduced MAPK activation by GnRH-a and TPA, but had no effect on GnRH-a and TPA stimulation of protein tyrosine phosphorylation. Interestingly, the removal of Ca2+ also partly inhibited the activation of MAPK by EGF and vanadate/H2O2. Thus, a calcium-dependent component(s) downstream of PKC and PTK might also participate in MAPK activation. Elevation of cAMP by forskolin exerted partial inhibition on EGF, but not on TPA or GnRH-a action, suggesting that MEK activators other than Raf-1 might be involved in GnRH action. We conclude that Ca2+, PTK, and PKC participate in the activation of MAPK by GnRH-a, with Ca2+ being necessary downstream to PKC and PTK.
Reiss
N
N
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.
Llevi
L N
LN
Shacham
S
S
Harris
D
D
Seger
R
R
Naor
Z
Z
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D010766:Phosphorylation; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000201:enzymology*; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D017329:Triptorelin Pamoate; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007527:Isoenzymes; D010566:Virulence Factors, Bordetella; D017329:Triptorelin Pamoate; D007987:Gonadotropin-Releasing Hormone; D015759:Ionomycin; D000242:Cyclic AMP; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium