Identification of a C-terminal cdc25 sequence required for promotion of germinal vesicle breakdown.
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.
Powers
E A
EA
Cell and Molecular Biology, Pharmacia and Upjohn, Inc., Kalamazoo, MI 49007, USA.
Thompson
D P
DP
Garner-Hamrick
P A
PA
He
W
W
Yem
A W
AW
Bannow
C A
CA
Staples
D J
DJ
Waszak
G A
GA
Smith
C W
CW
Deibel
M R
MR
Fisher
C
C
D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D017124:Conserved Sequence / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D008666:Metalloendopeptidases / Q000378:metabolism; D008845:Microinjections; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D014982:Xenopus laevis; D020687:cdc25 Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D018797:Cell Cycle Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016203:CDC2 Protein Kinase; D020687:cdc25 Phosphatases; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase