title: Gonadotropin-releasing hormone and protein kinase C signaling to ERK: spatiotemporal regulation of ERK by docking domains and dual-specificity phosphatases.
abstract: Activated ERK translocates to the nucleus to regulate transcription. Spatiotemporal aspects of this response dictate biological consequences and are influenced by dual-specificity phosphatases (DUSPs) that can scaffold and dephosphorylate ERK. In HeLa cells, GnRH causes transient and protein kinase C (PKC)-dependent ERK activation, but termination mechanisms are unknown. We now explore DUSP roles using short inhibitory RNA to knock down endogenous ERK, adenoviruses to express GnRH receptors and add-back ERK2-GFP, and automated microscopy to monitor ERK location and activation. GnRH caused rapid and transient increases in dual phosphorylated ERK2 (ppERK2) and nuclear to cytoplasmic ERK2-green fluorescent protein (GFP) ratio, whereas responses to a PKC-activating phorbol ester were more sustained. In cells expressing D319N ERK2-GFP (D319N mutation impairs docking-domain-dependent binding to DUSPs), GnRH caused more sustained increases in ppERK2 and nuclear to cytoplasmic ERK2-GFP ratio and also had more pronounced effects on Egr-1 luciferase (a transcriptional reporter for ERK activation). Cycloheximide caused more sustained effects of GnRH and phorbol ester on ppERK, suggesting termination by nuclear-inducible DUSPs. GnRH also increased expression of nuclear-inducible DUSP1 and -4, but their knockdown did not alter GnRH-mediated ERK signaling. Screening a short inhibitory RNA library targeting 16 DUSPs (nuclear-inducible DUSPs, cytoplasmic ERK MAPK phosphatases, c-Jun N-terminal kinase/p38 MAPK phosphatases, and atypical DUSPs) revealed GnRH effects to be influenced by DUSPs 5, 9, 10, 16, and 3 (i.e. by each DUSP class). Thus, GnRH-mediated ERK responses (like PKC-mediated ERK responses) are dependent on protein neosynthesis and docking-domain-dependent binding, but for GnRH activation (unlike PKC activation), this does not reflect dependence on nuclear-inducible DUSPs. Termination of these GnRH effects is apparently dependent upon a preexisting rapid turnover protein.
authors.affiliation: Department of Clinical Sciences at South Bristol, University of Bristol, Bristol BS1 3NY, UK.
authors.forename: Stephen Paul
authors.initials: SP
authors.lastname: Armstrong
authors.forename: Christopher James
authors.initials: CJ
authors.lastname: Caunt
authors.forename: Craig Alexander
authors.initials: CA
authors.lastname: McArdle
mesh_terms: D000818:Animals; D054637:Dual-Specificity Phosphatases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D055785:Gene Knockdown Techniques; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D007987:Gonadotropin-Releasing Hormone; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D054637:Dual-Specificity Phosphatases; D013755:Tetradecanoylphorbol Acetate