GnRH acts via G-protein coupled receptors to stimulate phospholipase C. This activates protein kinases C, driving the activation of extracellular signal regulated kinases (ERKs) that mediates transcriptional effects of GnRH. GnRH is secreted in pulses that cause rapid, transient and reproducible ERK activation (1). ERK response kinetics dictate biological consequences in many systems and, although GnRH-mediated ERK activation has been thoroughly explored, little is known about inactivation mechanisms. Here, we have tested how protein phosphatases shape acute ERK responses, using automated microscopy to monitor levels of ppERK1/2 (dual phosphorylated ERK 1/2). GnRH rapidly increased ppERK1/2 (max. at 6 min in HeLa cells transduced with mouse GnRH receptors), an effect that was rapidly reversed by MEK inhibition (PD184352) at 6 min. This effect (half-time 1.43 min, K 0.48±0.05/min) provides a measure for the ppERK1/2 dephosphorylation rate. The half-time was increased 61% by sodium orthovanadate (broad spectrum tyrosine phosphatase inhibitor), 39% by okadaic acid (protein ser/thr phosphatase 1, 2A and 2B inhibitor) and was unaltered by cyclosporine A (protein ser/thr phosphatase 2B inhibitor). In similar experiments with cells expressing wild-type or mutant ERK2-GFP reporters the inactivation half-time was increased >2 fold in cells with D319N-ERK2 (a mutation that prevents D-domain dependent binding of ERK to substrates and phosphatases) but not altered in cells with Y261A-ERK2 (that prevents DEF-domain-dependent binding) or K52R-ERK2 (that prevents ERK kinase activity). It was also unaltered by siRNA-mediated knock-down of the nuclear-inducible MAPK phosphatases (MKPs), the cytoplasmic ERK-directed MKPs, the JNK/p38 selective MKPs or the catalytic subunits of protein phosphatase 2A. Sodium orthovanadate also increased the ERK inactivation rate in LβT2 cells (gonadotroph lineage cells with murine GnRH receptors). Thus, although the mechanisms terminating GnRH-mediated ERK activation remain unknown, the process likely involves D-domain dependent association with vanadate sensitive tyrosine phosphatase(s). 1. Armstrong SP et al. (2010) J Biol Chem 285:2436071.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.