The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1.
The ordered progression through the cell cycle depends on regulating the abundance of several proteins through ubiquitin-mediated proteolysis. Degradation is precisely timed and specific. One key component of the degradation system, the anaphase promoting complex (APC), is a ubiquitin protein ligase. It is activated both during mitosis and late in mitosis/G(1), by the WD repeat proteins Cdc20 and Cdh1, respectively. These activators target distinct sets of substrates. Cdc20-APC requires a well-defined destruction box (D box), whereas Cdh1-APC confers a different and as yet unidentified specificity. We have determined the sequence specificity for Cdh1-APC using two assays, ubiquitination in a completely defined and purified system and degradation promoted by Cdh1-APC in Xenopus extracts. Cdc20 is itself a Cdh1-APC substrate. Vertebrate Cdc20 lacks a D box and therefore is recognized by Cdh1-APC through a different sequence. By analysis of Cdc20 as a substrate, we have identified a new recognition signal. This signal, composed of K-E-N, serves as a general targeting signal for Cdh1-APC. Like the D box, it is transposable to other proteins. Using the KEN box as a template, we have identified cell cycle genes Nek2 and B99 as additional Cdh1-APC substrates. Mutation in the KEN box stabilizes all three proteins against ubiquitination and degradation.
Pfleger
C M
CM
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 USA.
Kirschner
M W
MW
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D001665:Binding Sites; D064199:Cdc20 Proteins; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D019926:Cyclin B / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014981:Xenopus
C577495:CDC20 protein, S cerevisiae; C577583:CDH1 protein, S cerevisiae; D064199:Cdc20 Proteins; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins; D019926:Cyclin B; D005656:Fungal Proteins; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases