Raf-1 kinase, a potential regulator of intracellular pH in Xenopus oocytes

Abstract

Xenopus laevis oocytes undergo an increase in intracellular pH (pH i) from 7.2 to 7.7 due to the upregulation of Na + H + antiporters in their plasma membrane during oocyte meiotic maturation. Up-regulation of Na + H + exchangers (NHE) found in other cell systems appears to be controlled, in some cases, by direct phosphorylation of the exchanger. A number of active protein kinases can be found in maturing Xenopus oocytes. These include, c-mos kinase, Raf-1 kinase, mitogen-activated kinase kinase (MAPKK), MAPK, ribosomal S6 kinase (RSK), and histone H-1 kinase. Our previous study indicated that c-mos kinase, was involved in regulating the increase in oocyte pH i. In the current study, we show that when mRNA coding for a constitutively active form of Raf-1 kinase ( ΔN- Raf-1) was microinjected into oocytes, the protein product induced an increase in oocyte pH i. On the contrary, the injection of mRNA coding for wild-type Raf-1 (WT-Raf-1) or a kinase-deficient form of Raf-1 (KD-Raf-1) had no effect on the recipient oocyte pH i. 8-Br-cAMP and forskolin blocked the increase in pH i during oocyte meiotic maturation, but had no effect on the Raf-1-induced increase in oocyte pH i. Studies using antisense c-mos oligos demonstrated that Raf-1 was not working via a feedback loop to endogenous c-mos mRNA within the recipient oocytes. Experiments using the selective MAPKK inhibitor, PD 98059, indicated that the Raf-1 effect on oocyte pH i was not mediated by the downstream kinase, MAPKK. Therefore, Raf-1 appears to act independently of c-mos kinase in a pathway, not involving MAPKK, leading to the up-regulation of the Na + H + antiporters in Xenopus oocytes.