Gonadotropin-releasing hormone and protein kinase C signaling to ERK: spatiotemporal regulation of ERK by docking domains and dual-specificity phosphatases

Mol Endocrinol. 2009 Apr;23(4):510-9. doi: 10.1210/me.2008-0333. Epub 2009 Jan 29.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dual-Specificity Phosphatases / genetics
  • Dual-Specificity Phosphatases / metabolism*
  • Enzyme Activation
  • Gene Knockdown Techniques
  • Gonadotropin-Releasing Hormone / metabolism*
  • HeLa Cells
  • Humans
  • Mitogen-Activated Protein Kinase 1 / genetics
  • Mitogen-Activated Protein Kinase 1 / metabolism*
  • Mitogen-Activated Protein Kinase 3 / genetics
  • Mitogen-Activated Protein Kinase 3 / metabolism*
  • Protein Kinase C / metabolism*
  • Protein Structure, Tertiary
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction / physiology*
  • Tetradecanoylphorbol Acetate / metabolism

Substances

  • RNA, Small Interfering
  • Recombinant Fusion Proteins
  • Gonadotropin-Releasing Hormone
  • Protein Kinase C
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Dual-Specificity Phosphatases
  • Tetradecanoylphorbol Acetate