Mechanisms of GnRH-induced extracellular signal-regulated kinase nuclear localization.
Gonadotropin-releasing hormone receptors (GnRHR) mediate activation and nuclear translocation of the extracellular signal regulated kinases 1 and 2 (ERK) by phosphorylation on the TEY motif. This is necessary for GnRH to initiate transcriptional programmes controlling fertility, but mechanisms that govern ERK targeting are unclear. Using automated microscopy to explore ERK regulation in single cells, we find that GnRHR activation induces marked redistribution of ERK to the nucleus and that this effect can be uncoupled from the level of TEY phosphorylation of ERK. Thus, 5 min stimulation with 100 nM GnRH increased phospho-ERK levels (from 89 ± 34 to 555 ± 45 arbitrary fluorescence units) and increased the nuclear:cytoplasmic (N:C) ERK ratio (from 1.36 ± 0.06 to 2.16 ± 0.05) in the whole cell population, but it also significantly increased N:C ERK in cells binned according to phospho-ERK levels. This phosphorylation unattributable component of the ERK translocation response occurs at a broad range of GnRHR expression levels, in the presence of tyrosine phosphatase and protein synthesis inhibitors, and in ERK mutants unable to undergo catalytic activation. It also occurred in mutants incapable of binding the DEF (docking site for ERK, F/Y-X-F/Y-P) domains found in many ERK binding partners. It was however, reduced by MEK or PKC inhibition and by mutations preventing TEY phosphorylation or that abrogate ERK binding to D (docking) domain partners. We therefore show that TEY phosphorylation of ERK is necessary, but not sufficient for the full nuclear localization response. We further show that this "phosphorylation unattributable" component of GnRH-mediated ERK nuclear translocation requires both PKC activity and association with partner proteins via the D-domain.
Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom.
Christopher J
CJ
Caunt
Rebecca M
RM
Perett
Robert C
RC
Fowkes
Craig A
CA
McArdle
D020816:Amino Acid Motifs; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; 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*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011966:Receptors, LHRH / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D059010:Single-Cell Analysis
D004791:Enzyme Inhibitors; C484844:GNRHR protein, human; D011500:Protein Synthesis Inhibitors; D011966:Receptors, LHRH; D011493:Protein Kinase C; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3