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Cell Cycle-Regulated Recognition of the Destruction Box of Cyclin B by the APC/C in Xenopus Egg Extracts

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Cell Cycle-Regulated Recognition of the Destruction Box of Cyclin B by the APC/C in Xenopus Egg Extracts Molecular Cell, Vol. 13, 137–147, January 16, 2004, Copyright 2004 by Cell Press Cell Cycle-Regulated Recognition of the Destruction Box of Cyclin B by the APC/C in Xenopus Egg Extracts Hiroyuki Yamano,1 Julian Gannon, Hiro Mahbubani, ubiquitylates other proteins during the cell cycle, such and Tim Hunt* as Securin/Cut2/Pds1, Nek2A, Xkid1, Cdc20, Cdc5, Cancer Research UK Ase1, and Geminin and targets them for destruction by Clare Hall Laboratories the 26S proteasome (Charles et al., 1998; Cohen-Fix et South Mimms al., 1996; Funabiki and Murray, 2000; Funabiki et al., Herts EN6 3LD 1996; Hames et al., 2001; Juang et al., 1997; McGarry United Kingdom and Kirschner, 1998; Shirayama et al., 1998). The APC/C thus regulates at least two key events in mitosis: sister chromatid separation and the inactivation of CDKs Summary (cyclin-dependent kinases) to exit from mitosis (Harper et al., 2002; Morgan, 1999; Peters, 2002; Zachariae and Substrates for mitotic proteolysis such as cyclin B Nasmyth, 1999). This ordered ubiquitylation/proteolysis have a 9 residue destruction motif, the destruction box is regulated by activation of the APC/C via phosphoryla- (D-box). To identify the receptor that specifically binds tion of its subunits (Golan et al., 2002; Kotani et al., the D-box, we used affinity chromatography with im- 1998; Lahav-Baratz et al., 1995; Patra and Dunphy, 1998; mobilized D-box matrices. We find that the APC/C Rudner and Murray, 2000; Yamada et al., 1997) and also from Xenopus egg extracts binds to the D-box of cyclin by the recruitment of activators, comprising two related B, whereas Fizzy (Cdc20) does not. Mutations in the WD40-repeat proteins, Fizzy (Cdc20) and Fizzy-related D-box abolished this interaction. We show that this (Cdh1/Hct1). Genetic experiments have demonstrated binding is regulated in the cell cycle, such that the that the Fizzy family proteins are essential for the APC/ APC/C from egg extracts in interphase does not bind C-dependent ubiquitylation, and that they somehow or- to the D-box matrix. Our results suggest that the chestrate temporal differences in substrate ubiquityla- APC/C forms a stable interaction with the D-box of its tion during the progression of mitosis and G1-phase substrates in a cell cycle-dependent manner. (Dawson et al., 1995; Fang et al., 1998; Kramer et al., 2000; Lim et al., 1998; Schwab et al., 1997; Shirayama Introduction et al., 1998; Sigrist et al., 1995; Sigrist and Lehner, 1997; Visintin et al., 1997; Zachariae et al., 1998). Nonetheless, The ubiquitin pathway is an ATP-dependent tagging sys- the roles of Fizzy family proteins for the APC/C-depen- dent proteolysis are not well defined. The favored cur- tem for protein degradation. It is an essential system in rent model for the role of Fizzy family proteins (Cdc20, eukaryotic cells, where it is used for degrading damaged Cdh1/Hct1) is to recognize the substrates (Burton and and misfolded proteins and also for degrading short- Solomon, 2001; Hilioti et al., 2001; Ohtoshi et al., 2000; lived regulatory proteins during processes such as the Pfleger et al., 2001; Schwab et al., 2001) and, presum- cell cycle, transcription, signal transduction, and devel- ably, deliver them to the APC/C, although they might opment (Hershko and Ciechanover, 1998; Jackson et have additional roles in the regulation of APC-dependent al., 2000; Peters, 2002; Zachariae and Nasmyth, 1999). proteolysis. On the other hand, two recently published The covalent attachment of ubiquitin chains to proteins reports claim that the APC/C, rather than Fizzy, is in- can be highly selective and delicately regulated. It is volved in substrate recognition (Meyn et al., 2002; Pass- achieved by the sequential action of three enzymes, E1, more et al., 2003). Thus, it has become less clear how E2, and E3; ubiquitin is first attached by a thioester APC/C substrates are recognized, and by what. Notably, bond to a ubiquitin-activating enzyme (E1) in an ATP- those APC/C substrates that are destroyed in anaphase, dependent manner, then transferred to a small and with no exception, have a destruction box (D-box) as somewhat specific ubiquitin-carrying protein (E2), and their destruction signal. Thus, it is important to find what finally the ubiquitin is conjugated onto the target sub- binds to the D-box and how the recognition is coordi- strates either by E2 alone or, more often, in conjunction nated with ubiquitylation. We may ask, for example, with a substrate-specific ubiquitin ligase (E3). Proteins whether it is the recognition or the ubiquitylation that is carrying long chains or trees of ubiquitin are then deliv- regulated? Here, we directly explore such questions in ered to the proteasome for degradation (Hershko and Xenopus egg extracts, where the picture is simplified Ciechanover, 1998; Hochstrasser, 1996; Varshavsky, by the absence of Fizzy-related (Lorca et al., 1998). We 1997). Since this proteolysis pathway is a precise and used affinity chromatography with immobilized D-box irreversible process, it allows a rapid switch to control matrices to search for the D-box “receptor.” In frog egg biological transitions. The anaphase-promoting com- extracts we find that very little Fizzy binds to the D-box, plex/cyclosome (APC/C) is a large E3 ubiquitin ligase whereas most of the APC/C is specifically retained by complex, which was originally identified as cyclin B ubi- a D-box matrix. We also present data suggesting that quitin ligase (Irniger et al., 1995; King et al., 1995; Su- purified APC/C can bind to the D-box matrix without dakin et al., 1995; Tugendreich et al., 1995), but it also Fizzy protein. Moreover, the interaction between the D-box and the APC/C appears to be regulated in the *Correspondence: [email protected] cell cycle. Although these experiments do not define 1Present address: Marie Curie Research Institute, The Chart, Oxted, the role of Fizzy, our data imply that the APC/C can bind Surrey RH8 0TL, United Kingdom. to the D-box without Fizzy protein, once it has been Molecular Cell 138 Figure 1. Affinity Chromatography with Im- mobilized D-Boxes (A) Schematic diagram of the D-box affinity columns; wild-type D-boxes are indicated by open and mutant D-boxes by filled rect- angles. (B) Flow-through of the various D-box col- umns were used for testing cyclin proteolysis in frog egg extracts. Full-length fission yeast cyclin B (Cdc13) and a version lacking 67 N-terminal residues (⌬67, stable control) were used as substrates. CaCl2 was added to initi- ate proteolysis. activated in mitosis, a process that somehow involves even in the presence of Ca2ϩ, whereas the flow-through Fizzy. from the column carrying the mutated tandem D-box efficiently destroyed Cdc13. The single N70 affinity resin Results was slightly less effective than N70-2X, although its FT showed impaired destruction for Cdc13 compared to Destruction Box Affinity Columns Deplete Essential the FT from the mutated D-box column (Dm) or GST Elements of Cyclin Proteolysis alone. These results indicate that the D-box affinity col- We constructed a variety of affinity columns to explore umns could deplete elements that are required for com- how destruction boxes (D-box) are recognized by the plete cyclin proteolysis. ubiquitylation machinery. Most experiments used the N-terminal 70 residues of fission yeast cyclin B (N70) D-Boxes Deplete APC/C Rather than Fizzy fused to GST, since we previously showed that this in Xenopus Egg Extracts N-terminal fragment allowed efficient APC/C-dependent Next, we investigated what factor(s) were depleted from ubiquitylation and proteolysis in Xenopus egg extracts egg extracts, rendering the extracts incompetent for (Yamano et al., 1996, 1998). Four constructs were made: cyclin destruction. Since we previously found that the N70 fused to GST, a tandem N70 construct (N70-2X), D-box receptor was immunoprecipitated by anti-Apc3 and the two equivalent constructs with the R and L of antibodies (Yamano et al., 1998), and there are several the D-box(es) mutated to A residues (Figure 1A). To test lines of evidence that the Fizzy family of proteins are whether these constructs worked as bait for the D-box involved in anaphase-promoting complex/cyclosome receptor, we used Xenopus egg extracts in which the (APC/C) substrate recognition (Burton and Solomon, APC/C-dependent cyclin destruction assay can be per- 2001; Hilioti et al., 2001; Ohtoshi et al., 2000; Pfleger et formed. Cytostatic factor (CSF) arrested Xenopus egg al., 2001; Schwab et al., 1997, 2001; Visintin et al., 1997), extracts are arrested at metaphase in meiosis II, and we examined which fractions contain the APC/C, Fizzy, addition of Ca2ϩ releases this arrest and triggers cyclin or Cdc2 (a negative control), after affinity chromatogra- proteolysis via APC/C activation (King et al., 1995; Masui phy. CSF-arrested metaphase egg extracts (input, I), and Markert, 1971; Murray et al., 1989; Tunquist and flow-through (F), and bound (B) fractions were immu- Maller, 2003; Watanabe et al., 1991; Yamano et al., 1998). noblotted with anti-Apc3, anti-Fizzy, or anti-Cdc2 anti- If essential elements such as a D-box receptor are de- bodies (Figure 2A). The flow-through from the tandem pleted by these D-box affinity columns, one would ex- N70 column contained only about 5% of the input pect that the flow-through (FT) would fail to destroy APC/C, indicating that the N70 column was able to de- cyclin even in the presence of Ca2ϩ. To assay D-box plete up to 95% of APC/C from egg extracts (lanes or D-box-independent cyclin proteolysis, we used two 1and 2).
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