ACTIVATION OF P90RSK DURING MEIOTIC MATURATION AND FIRST MITOSIS IN MOUSE OOCYTES AND EGGS - MAP KINASE-INDEPENDENT AND KINASE-DEPENDENT ACTIVATION

Mitogen-activated protein kinases (MAPK) become activated during the m eiotic maturation of oocytes from many species; however, their molecul ar targets remain unknown. This led us to characterize the activation of the ribosomal subunit S6 kinase of M(r) 82x10(3)-99x10(3) (p90(rsk) ; a major substrate of MAPK in somatic cells) in maturing mouse oocyte s and during the first cell cycle of the mouse embryo. We;assessed the phosphorylation state of p90(rsk) by examining the electrophoretic mo bility shifts on immunoblots and measured the kinase activity of immun oprecipitated p90(rsk) on a S6-derived peptide. Germinal vesicle stage (GV) oocytes contained a doublet of M(r) 82x10(3) and 84x10(3) with a low S6 peptide kinase activity (12% of the maximum level found in met aphase II oocytes). A band of M(r) 86x10(3) was first observed 30 minu tes after GV breakdown (GVBD) and became prominent within 2 to 3 hours , MAPK was not phosphorylated 1 hour after GVBD, when the p90(rsk)-spe cific S6 kinase activity reached 37% of the M II level. 2 hours after GVBD, MAPK became phosphorylated and p90(rsk) kinase activity reached 86% of the maximum level. The p90(rsk) band of M(r) 88x10(3), present in mature M II oocytes when 86 peptide kinase activity is maximum, app eared when MAPK phosphorylation was nearly complete (2.5 hours after G VBD), In activated eggs, the dephosphorylation of p90(rsk) to M(r) 86x 10(3) starts about 1 hour after the onset of pronuclei formation and c ontinues very slowly until the beginning of mitosis, when the doublet of M(r) 82x10(3) and 84x10(3) reappears. A role for a M-phase activate d kinase (like p34(cdc2)) in p90(rsk) activation was suggested by the reappearance of the M(r) 86x10(3) band during first mitosis and in 1-c ell embryos arrested in M phase by nocodazole, The requirement of MAPK for the full activation of p90(rsk) during meiosis was demonstrated b y the absence of the fully active M(r) 88x10(3) band in maturing c-mos (-/-) oocytes, where MAPK is not activated, The inhibition of kinase a ctivity in activated eggs by 6-DMAP after second polar body extrusion provided evidence that both MAPK- and p90(rsk)-specific phosphatases a re activated at approximately the same time prior to pronuclei formati on.