Inhibition of Mitogen-Activated Protein Kinase Activity by Prolactin Signaling Through the Short Form of Its Receptor: Involvement of Dual Specific Protein Phosphatase 27

Y. SANGEETA DEVI, ANITA SEIBOLD , AURORA SHEHU, JULIA HALPERIN, JAMIE LE, EVELYN MAIZELS, AND GEULA GIBORI

Pittsburgh. USA

Congreso; Society for the Study of Reproduction 42nd Annual Meeting; 2009

Mitogen activated protein kinase (MAPK) has been implicatedin the regulation of granulosa cell differentiation, steroidogenicgene expression and meiotic arrest of oocytes. Previously, wehave shown that prolactin (PRL) signaling in the transgenicmice expressing only short isoform of prolactin receptor (PRL-PR)suffer from early follicular activation followed by massivegranulosa and oocyte cell death and premature ovarian failure.To determine whether MAPK pathway is involved in these processes,pseudopregnant mice expressing only PRL-RS were treated withPRL for different time points. We found transient and time dependantinhibition of ERK1/2 activity in the ovary. The phosphorylationof ELK-1 and p90RSK, downstream targets of ERK1/2, were alsoseverely inhibited by PRL in the ovary of these mice. However,the phosphorylation status of immediate upstream kinase MEK1/2that phosphorylates ERK1/2 was not affected by PRL suggestinginvolvement of MAPK specific phosphatase(s) in the dephosphorylationof ERK1/2. To determine the phosphatase responsible for PRLmediated ERK1/2 inhibition, we examined the expression levelsof immediate early genes Map Kinase Phosphatase (MKP)1 and MKP5in the ovary of wild type and PRL-RS expressing mice. Surprisingly,expression levels of both MKP1 and MPK5 were barely detectableand much lower in the PRL-RS expressing ovaries as comparedto wild type ovaries, suggesting that these phosphatases arenot involved in PRL mediated ERK1/2 inhibition. We further examinedPRL/PRL-RS mediated inhibition of MAPK activity in GG-CL cells,a PRL responsive ovarian derived cell line. GGCL cells transfectedwith PRL-RS and treated with PRL showed remarkable inhibitionof ERK1/2 in the same pattern as that seen in vivo, while MEK1/2remain unchanged. The inhibition of MAPK was independent ofJak2 activity, since AG490 treatment had no apparent effect.To our surprise, we found that none of the serine phosphatases(PP2A, PP2B and PP1) or the protein tyrosine phosphatases (1Bor SHP2) appears to be involved in PRL/PRL-RS mediated inhibitionof ERK1/2 activity, as shown by inhibitor studies. To have abetter understanding of what signaling molecules are activatedthrough PRL-RS, which may have a role in MAPK regulation, weperformed a GST pull down assay with PRL-RS fusion protein taggedwith GST. A protein band of approximately 30Kda was found tobe associated with PRL-RS. MALDI-TOF analysis revealed the identityof this protein as dual specific phosphatase 27 (DUSP27). Interestingly,PRL mediated ERK1/2 inhibition was reversed in GGCL cells transfectedwith PRL-RS by NSC 95397 and NSC663284, a new class of dualspecific phosphatase inhibitors. Immunoprecipitation and Westernanalysis further showed physical association of ERK1/2 withDUSP27 suggesting that DUSP27 is a strong candidate for PRLmediated inhibition of ERK1/2. Taken together, our resultsindicate that PRL mediated dephosphorylation of ERK1/2 throughPRL-RS markedely inhibits MAPK activity contributing to earlyfollicular activation and massive cell death. Our results alsoreveal, for the first time, the novel association of DUSP27phosphatase, with the PRL-RS and ERK1/2 and suggest that PRLmediated dephosphorylation of ERK1/2 involves this phosphatase.Supported by NIH HD12356 and HD11119 (GG), T32 HL007692 (JL& AS) and APS fellowship (JH)