ACTIVATION OF AN 85 KDA RIBOSOMAL S6 KINASE DURING SEROTONIN-INDUCED OOCYTE MATURATION

Oocytes from the Japanese clam Ruditapes philippinarum are naturally b locked at the prophase-I stage of meiosis. Following physiological act ivation by the neurohormone serotonin (5HT), oocytes undergo germinal vesicle breakdown (GVBD) and reach a second cell cycle arrest in metap hase-I. To identify the kinases activated during meiosis reinitiation, we used a phosphorylation assay following sodium dodecyl sulphate-pol yacrylamide gel electrophoresis and in situ renaturation. A soluble 85 -kDa serine/threonine kinase (PK85) was highly and consistently activa ted (up to 17-fold) within 5 minutes following addition of the hormone . This activation occurred 5 to 10 minutes before GVBD and only when 5 HT concentration was sufficient to induce meiosis reinitiation. The ca lcium ionophore A23187 and NH4Cl, two compounds known to induce GVBD b y increasing intracellular calcium concentration, also activate PK85. In crude oocyte extracts, the presence of beta-glycerophosphate, NaF, okadaic acid, calyculin A or microcystin, prevented inactivation of PK 85, suggesting that it is activated by phosphorylation. Partial purifi cation of PK85 followed by Western blotting showed that this kinase is related to the ribosomal S6 kinase pp90(rsk). PK85 phosphorylates the peptides LRRASLG (kemptide) and PLARTLSVAGLPGGK (syntide-2), and to a lesser extent the synthetic polyamino acids poly(R(3):S-1) while myel in basic protein (MBP), histone III-S, casein, the peptides pEKRPSQRSK YL ((pGlu(4))-MBP 4-14), GTFRASIRRLAARRR (NIMA kinase substrate), the protein kinase C (PKC) substrate LRTLRR and the synthetic polyaminoaci ds poly(R(1):P-1:T-1) were poor substrates. 5HT-induced GVBD and PK85 activation are both inhibited by the phorbol ester 12-myristate 13-ace tate (PMA) and this inhibition can be reversed by 5 mu M of the bisind olylmaleimide GF109203X, a potent PKC inhibitor. PMA inhibitory action appears to take place between 5HT binding to its receptor and the int racellular calcium surge since it has no effect on GVBD induced by cal cium ionophore A23187 and thapsigargin. Taken together, these results suggest that serotonin-induced activation of PK85 occurs after the int racellular calcium surge in a PKC-independent pathway.