Cdk1 Is Essential for Mammalian Cyclosome/APC Regulation Tamar Listovsky, Amit Zor, Ayelet Laronne, and Michael Brandeis 1 Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel The cyclosome/APC (anaphase-promoting complex), the major component of cell-cycle-specific ubiquitin- mediated proteolysis of mitotic cyclins and of other cell cycle proteins, is essential for sister chromatid separation and for exit from mitosis. Cyclosome activ- ity and substrate specificity are modulated by phos- phorylation and by transient interactions with Fizzy/ cdc20 (Fzy) and Fizzy-related/Hct1/Cdh1 (Fzr). This regulation has been studied so far in Drosophila em- bryos, in yeast, and in cell-free extracts in vitro. Study- ing cyclosome regulation in mammalian cells in vivo we found that both Fzr overexpression and Cdk1 inhi- bition can override the prometaphase checkpoint. We further show that Fzr activation of the cyclosome is negatively regulated by Cdk1. Finally, we show that the mammalian cdc14 phosphatase, like its budding yeast homologue, plays a role in cyclosome pathway regulation. These results suggest that Cdk1 is essen- tial for coupling various activities of the cyclosome and in particular for preventing Fzr from short-cir- cuiting the spindle pole checkpoint. Cdk1– cyclin B is thus an inhibitor, activator, and substrate of the cy- closome. © 2000 Academic Press Key Words: Fizzy; Fizzy-related; Hct1; Cdc20; cyclin B; cdc14. INTRODUCTION Ubiquitin-mediated proteolysis plays a key role in the regulation of the eukaryotic cell cycle. Mitotic cyc- lins, as well as other cell cycle regulators, are degraded by this pathway specifically during mitosis and G1 phase. Cell cycle-controlled ubiquitination and subse- quent proteolysis of these substrates requires the ac- tivity of a specific ubiquitin carrier protein–E2–C/ UbcH10 [1, 2], as well as a large complex called cyclosome or APC (anaphase promoting complex) [3, 4], which serves as the ubiquitin ligase (E3). While UbcH10 is constitutively active, the cyclosome exhibits a complex cell-cycle-dependent pattern of activity. This pattern is generated by reversible phosphorylation of several of the cyclosome subunits [5–7], as well as by transient association with two WD-repeat proteins— Fizzy/p55 cdc /Cdc20/Slp1 (Fzy) and Fizzy-related/Cdh1/ Hct1/Ste9 (Fzr) [8 –16]. Cyclosome activation by Fzy occurs during the early stages of mitosis and depends on cyclosome phosphor- ylation by Cdk1 [7]. In yeast, as well as in metazoa, the Fzy– cyclosome complex is required for the proteolysis of Pds1 [17, 18] an essential prerequisite of sister chro- matid separation [19]. Fzy– cyclosome activity is also responsible, at least partially, for the degradation of mitotic cyclins [8]. Tight inhibition of the Fzy– cyclo- some complex by the spindle pole checkpoint mecha- nism prevents premature sister chromatid separation both in yeast [20] and in metazoa [21, 22]. Moreover, Fzy has been found to associate and to interact with a variety of checkpoint proteins [20 –23]. Fzy activation of the cyclosome is a crucial but rela- tively short event lasting probably only several min- utes each cycle. During the rapid early embryonic cell cycles it seems to be the sole activator of the cyclosome [8, 12]. The activation of the cyclosome by Fzr, in con- trast, observed in yeast and in somatic cells, seems to last about half a cell cycle and is much less understood. This activation is believed to be initiated at anaphase and to partially drive the degradation of B-type cyclins and probably of other, yet to be identified, substrates. Fzr is bound to the cyclosome throughout G1 of the somatic cell cycle and is probably responsible for the G1-specific cyclosome activity observed in these cells [24]. In cell extracts, Fzr is capable of activating both mitotic and interphase cyclosome [13, 14], while Fzy is capable of activating only the phosphorylated mitotic cyclosome [7]. In contrast to the oscillating Fzy [25], Fzr is present throughout the cell cycle [14]. The bind- ing of Hct1, the Saccharomyces cerevisiae homologue of Fzr, to the cyclosome, is negatively regulated by phos- phorylation by Cdk1, the only cyclin-dependant kinase in budding yeast [26, 27]. We have studied the regulation of cyclosome-specific proteolysis in mammalian cells in vivo. We found that Fzr overexpression, as well as Cdk1 inhibition, is ca- pable of overriding the spindle pole checkpoint and of activating the cyclosome in prometaphase-arrested cells. We show that Cdk1 inhibition enabled Fzr bind- ing and activation of the prometaphase cyclosome. We 1 To whom reprint requests should be addressed. Fax: 972-2- 6586975. E-mail: brandeis@leonardo.ls.huji.ac.il. 184 0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Experimental Cell Research 255, 184 –191 (2000) doi:10.1006/excr.1999.4788, available online at http://www.idealibrary.com on