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

ABSTRACT

Glutathione S-transferase (GST)-cdc25B(31-566) induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. Purified, N-terminally truncated forms of cdc25B did not induce GVBD, even though many had phosphatase activity and activated cdc2 in vitro. N-terminally truncated forms of cdc25B inhibited induction of GVBD by longer forms of the enzyme suggesting 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 determine the region of peptide sequence that was inhibitory, cdc25B(356-556) was subjected to proteolysis with endoproteinase lys-C. Following a demonstration that the resulting peptide mixture inhibited GST-cdc25B-dependent GVBD, a series of peptides spanning amino acids at the C-terminus were synthesized. The peptide TRSWAGERSR inhibited GVBD induced by GST-cdc25B. An alanine scan of the peptide revealed residues critical for GVBD inhibition, and site-directed mutagenesis of the corresponding residues in GST-cdc25B(31-566) eliminated its ability to induce GVBD. These results demonstrate that a cdc25B C-terminal domain, involved in dominant-negative inhibition of GVBD-competent cdc25B, is required for induction of GVBD following microinjection into oocytes.