Fig. 6.
The PI3K–Akt pathway is not sufficient for phosphorylation of Akt substrates, including Cdc25 and Myt1, but the atypical Gβγ pathway enhances phosphorylation even in the absence of cyclin-B–Cdk1-dependent negative feedback. (A) Hypothesis tested in B–H. (B–D) PH–GFP was expressed in immature oocytes, followed by expression of either FLAG–CA-PI3K or Gβγ through incubation with roscovitine for 5 h after mRNA injection. For treatment with 1-MeAde, immature oocytes were pre-incubated with roscovitine, then treated with a supra-threshold concentration of 1-MeAde for 4 min. The oocytes were analyzed by immunoblotting (B) or confocal laser microscopy (C). In all cases, each oocyte had an intact germinal vesicle, within which some PH–GFP had accumulated, although in some cases it was out of the focal plane. Scale bar: 50 μm. The plasma-membrane-to-cytoplasm ratio of fluorescence density is shown in D. Data represent mean values±s.d. (five oocytes for immature and Gβγ, six oocytes for CA-PI3K). P-values (one-tailed t-test): *1, P<10−8; *2, P<10−3. (E,F) After treatment with a supra-threshold concentration of 1-MeAde (500 nM), or FLAG–CA-PI3K or Gβγ expression with roscovitine, oocytes were analyzed by immunoblotting (E). Phosphorylation levels relative to those after treatment with 1-MeAde were quantified. Data represent mean values±s.d. from three independent experiments (F). P-values (one-tailed t-test) for Akt: *1, *5, *6, P>0.05 (not significant, n.s.); *4, P<10−3; *2, *3, P<10−4. For Cdc25, *3, P<0.05; *1, P<0.01; *5, P<10−3; *2, *4, *6, P<10−4. (G,H) Immature oocytes were injected with the GST-Myt1-S75-peptide, followed by expression of either CA-PI3K or Gβγ by mRNA-injection, then incubated with roscovitine for 5 h. For 1-MeAde treatment, oocytes were treated with a supra-threshold (500 nM) concentration of 1-MeAde for 4 min after pre-incubation with roscovitine for 5 h. The oocytes were analyzed by immunoblotting (G). Phosphorylation levels relative to those after treatment with 1-MeAde were quantified. Data represent mean values±s.d. from three independent experiments (H). In F and H, the numbers showing comparisons (*1 to *6) apply to both the upper and the lower panels. P-values (one-tailed t-test) for Akt: *1, *5, *6, P>0.05 (n.s.); *2, *3, P<0.01; *4, P<10−5. For the Myt1-S75-peptide (S75-pep): *1, P>0.05 (n.s.); *5, *6, P<0.05; *2, *3, P<0.01; *4, P<10−4.