Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates.
Mitotic cyclins are abruptly degraded at the end of mitosis by a cell-cycle-regulated ubiquitin-dependent proteolytic system. To understand how cyclin is recognized for ubiquitin conjugation, we have performed a mutagenic analysis of the destruction signal of mitotic cyclins. We demonstrate that an N-terminal cyclin B segment as short as 27 residues, containing the 9-amino-acid destruction box, is sufficient to destabilize a heterologous protein in mitotic Xenopus extracts. Each of the three highly conserved residues of the cyclin B destruction box is essential for ubiquitination and subsequent degradation. Although an intact destruction box is essential for the degradation of both A- and B-type cyclins, we find that the Xenopus cyclin A1 destruction box cannot functionally substitute for its B-type counterpart, because it does not contain the highly conserved asparagine necessary for cyclin B proteolysis. Physical analysis of ubiquitinated cyclin B intermediates demonstrates that multiple lysine residues function as ubiquitin acceptor sites, and mutagenic studies indicate that no single lysine residue is essential for cyclin B degradation. This study defines the key residues of the destruction box that target cyclin for ubiquitination and suggests there are important differences in the way in which A- and B-type cyclins are recognized by the cyclin ubiquitination machinery.
King
R W
RW
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Glotzer
M
M
Kirschner
M W
MW
D000595:Amino Acid Sequence; D000705:Anaphase / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008938:Mitosis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012617:Sea Urchins; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014452:Ubiquitins / Q000378:metabolism*; D014981:Xenopus
D016213:Cyclins; D011994:Recombinant Proteins; D014452:Ubiquitins; D008239:Lysine