The KEN box: an APC recognition signal distinct from the D box targeted byCdh1

The ordered progression through the cell cycle depends on regulating the ab undance of several proteins through ubiquitin-mediated proteolysis. Degrada tion 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 r epeat proteins Cdc20 and Cdh1, respectively. These activators target distin ct sets of substrates. Cdc20-APC requires a well-defined destruction box (D box), whereas Cdh1-APC confers a different and as yet unidentified specifi city. We have determined the sequence specificity for Cdh1-APC using two as says, ubiquitination in a completely defined and purified system and degrad ation 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 Cd h1-APC through a different sequence. By analysis of Cdc20 as a substrate, w e 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 t ransposable to other proteins. Using the KEN box as a template, we have ide ntified cell cycle genes Nek2 and B99 as additional Cdh1-APC substrates. Mu tation in the KEN box stabilizes all three proteins against ubiquitination and degradation.