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

Xenopus laevis oocytes undergo an increase in intracellular pH (pHi) 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 pHi. 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 pHi. 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 pHi. 8‐Br‐cAMP and forskolin blocked the increase in pHi during oocyte meiotic maturation, but had no effect on the Raf‐1‐induced increase in oocyte pHi. 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 pHi 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.