Identification of a C-terminal cdc25 sequence required for promotion of germinal vesicle breakdown

Glutathione S-transferase (GST)-cdc25B(31-566) induced germinal vesicle bre akdown (GVBD) when microinjected into Xenopus oocytes. Purified, N-terminal ly truncated forms of cdc25B did not induce GVBD, even though many had phos phatase activity and activated cdc2 in vitro. N-terminally truncated forms of cdc25B inhibited induction of GVBD by longer forms of the enzyme suggest ing a direct interaction in vivo, cdc25B(356-556), but not cdc25B(364-529), inhibited GVBD induction by GST-cdc25B(31-566) suggesting that a region of cdc25B near to the C-terminus was responsible for the inhibition. To deter mine the region of peptide sequence that was inhibitory, cdc25B(356-556) wa s subjected to proteolysis with endoproteinase lys-C. Following a demonstra tion that the resulting peptide mixture inhibited GST-cdc25B-dependent GVBD , a series of peptides spanning amino acids at the C-terminus were synthesi zed. The peptide TRSWAGERSR inhibited GVBD induced by GST-cdc25B. An alanin e scan of the peptide revealed residues critical for GVBD inhibition, and s ite-directed mutagenesis of the corresponding residues in GST-cdc25B(31-566 ) eliminated its ability to induce GVBD. These results demonstrate that a c dc25B C-terminal domain, involved in dominant-negative inhibition of GVBD-c ompetent cdc25B, is required for induction of GVBD following microinjection into oocytes.