FIGURE 4.

Ringo/CDK and MAP kinase signaling pathways direct Musashi phosphorylation on Ser-322. A, MAP kinase signaling induces Musashi1 Ser-322 phosphorylation independently of CDK. Immature oocytes were injected with RNA encoding GST-tagged Musashi1 in the presence or absence of RNA encoding the CDK inhibitor Wee107 and incubated overnight. Oocytes were then split into pools and left untreated, injected with RNA encoding vRaf, or stimulated with progesterone as indicated. Progesterone- and vRaf-stimulated oocytes were segregated when the populations reached 50% GVBD (− and + GVBD, respectively). Protein lysates were analyzed by Western blot for Musashi1 phosphorylation on Ser-322, MAP kinase phosphorylation (indicative of activation), Cdc2 phosphorylation (indicative of inactive cyclin B/CDK), and relative GST-Musashi1 expression. B, progesterone-stimulated Musashi1 Ser-322 phosphorylation is mediated by both MAP kinase-dependent and MAP kinase-independent signaling. Immature oocytes were injected with RNA encoding GST-Musashi1 and incubated overnight, then treated with UO126 to inhibit MAP kinase signaling (+) or DMSO vehicle control (−) for 30 min before progesterone addition. Time-matched protein lysates were prepared when 50% of the control oocytes reached GVBD. Control oocytes were segregated based on whether they had (+) or had not (−) completed GVBD. Lysates were analyzed by Western blot for phosphorylation of Musashi1 on Ser-322, phosphorylation (activation) of MAP kinase, total MAP kinase, and expression of GST-Musashi1 in the same samples. C, shown is a schematic illustrating relevant progesterone-dependent signaling events impinging upon early class Musashi-mediated, mRNA translation before MPF (cyclin B/CDK) activation and oocyte GVBD. The points of experimental manipulation are shown along with the deduced Musashi amplification loops. For the sake of clarity, the complex roles of CPEB are not indicated in the diagram (“Discussion”).