Figure 3.
Phosphorylation of Xe-Cdc25A by ERK. (A) Conservation of consensus ERK phosphorylation motifs (Ser-Pro) in Xenopus and human Cdc25A proteins. (B) The indicated GST-fused Xe-Cdc25A peptides were incubated with [γ-32P]ATP and ERK protein and analyzed as described in Figure 2B. (C) GST-tagged full-length Xe-Cdc25A proteins (WT or S85A) were synthesized in wheat germ extracts, purified by GST-pull-down, incubated with either buffer (Cont.) or ERK protein, and analyzed by immunoblotting with anti-GST and anti-phospho-S85 antibodies. (D) Activated eggs were injected with 2 ng of mRNA encoding Myc-Xe-Cdc25A (WT or S85A), reinjected or not 2.5 h later with 9 ng of MEK-CA mRNA, and cultured for 50 min. Egg extracts were treated or not with λ-phosphatase and analyzed for Myc-Xe-Cdc25A and phospho-S85 by immunoblotting. (E) Activated eggs were injected with either buffer (Control), 18 ng of p21Cip1 mRNA, or 9 ng of MKP3 mRNA, reinjected 40 min later with 2 ng of Myc-Xe-Cdc25A mRNA, further injected 2.5 h later with 9 ng of MEK-CA mRNA, collected at the indicated times, and analyzed by immunoblotting as described in D. Four, three, four, and five independent experiments were performed for B, C, D, and E, respectively, and, for each, a typical result is shown.