Reiss N, Llevi L N, Shacham S, Harris D, Seger R, Naor Z
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.
Endocrinology. 1997 Apr;138(4):1673-82. doi: 10.1210/endo.138.4.5057.
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.
在促性腺激素细胞来源的αT3-1细胞系中研究了促性腺激素释放激素类似物[D-Trp6]GnRH（GnRH-a）刺激丝裂原活化蛋白激酶（MAPK，ERK）的机制。GnRH-a以及蛋白激酶C（PKC）激活剂12-O-十四酰佛波醇-13-乙酸酯（TPA）刺激了MAPK活性的持续反应，而表皮生长因子（EGF）刺激了短暂反应。MAPK激酶（MEK）也被GnRH-a以短暂的方式激活。如通过Mono-Q快速蛋白质液相色谱随后进行蛋白质印迹以及凝胶激酶测定所揭示的，GnRH-a和TPA显然主要激活了MAPK亚型ERK1。GnRH-a和TPA刺激了几种蛋白质的酪氨酸磷酸化，并且这种效应以及MAPK活性的刺激被PKC抑制剂GF 109203X抑制。同样，TPA敏感的PKC亚型的下调几乎消除了GnRH-a和TPA对MAPK活性的影响。此外，蛋白质酪氨酸激酶（PTK）抑制剂染料木黄酮抑制了蛋白质酪氨酸磷酸化，并使GnRH-a刺激的MAPK活性降低了50%，表明染料木黄酮敏感和不敏感的途径参与了GnRH-a的作用。尽管Ca2+离子载体只有轻微的刺激作用，但去除Ca2+显著降低了GnRH-a和TPA对MAPK的激活，但对GnRH-a和TPA刺激的蛋白质酪氨酸磷酸化没有影响。有趣的是，去除Ca2+也部分抑制了EGF和钒酸盐/H2O2对MAPK的激活作用。因此，PKC和PTK下游的钙依赖性成分也可能参与MAPK的激活。福斯可林升高cAMP对EGF的作用有部分抑制，但对TPA或GnRH-a的作用没有影响，表明除Raf-1之外的MEK激活剂可能参与了GnRH的作用。我们得出结论，Ca2+、PTK和PKC参与了GnRH-a对MAPK的激活，Ca2+在PKC和PTK的下游是必需的。
