Activation and nuclear translocation of PKCdelta, Pyk2 and ERK1/2 by gonadotropin releasing hormone in HEK293 cells.
The mechanism of agonist-induced activation of Pyk2 and its relationship with ERK1/2 phosphorylation was analyzed in HEK293 cells stably expressing the gonadotropin releasing hormone (GnRH) receptor. GnRH stimulation caused rapid and sustained phosphorylation of ERK1/2 and Pyk2 that was accompanied by their nuclear translocation. Pyk2 was also localized on cell membranes and at focal adhesions. Dominant negative Pyk2 (PKM) had no effect on GnRH-induced ERK1/2 phosphorylation and c-fos expression. These actions of GnRH on ERK1/2 and Pyk2 were mimicked by activation of protein kinase C (PKC) and were abolished by its inhibition. GnRH caused translocation of PKCalpha and delta, but not of epsilon, iota and lambda, to the cell membrane, as well as phosphorylation of Raf at Ser338, a major site in the activation of MEK/ERK1/2. Stimulation of HEK293 cells by EGF caused marked ERK1/2 phosphorylation that was attenuated by the selective EGFR receptor (EGF-R) kinase inhibitor, AG1478. However, GnRH-induced ERK1/2 activation was independent of EGF-R activation. These results indicate that activation of PKC is responsible for GnRH-induced phosphorylation of both ERK1/2 and Pyk2, and that Pyk2 activation does not contribute to GnRH signaling. Moreover, GnRH-induced phosphorylation of ERK1/2 and expression of c-fos in HEK293 cells is independent of Src and EGF-R transactivation, and is mediated through the PKC/Raf/MEK cascade.
Farshori
Parvaiz Q
PQ
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 6A-36, Bethesda, MD 20892-4510, USA.
Shah
Bukhtiar H
BH
Arora
Krishan K
KK
Martinez-Fuentes
Antonio
A
Catt
Kevin J
KJ
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D051418:Focal Adhesion Kinase 2; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D021381:Protein Transport / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011966:Receptors, LHRH / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D007295:Inositol Phosphates; D011966:Receptors, LHRH; D011994:Recombinant Proteins; D007987:Gonadotropin-Releasing Hormone; C497259:Prkcd protein, mouse; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases