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Regulation of B-Type Cyclin Proteolysis by Cdc28-Associated Kinases in Budding Yeast

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Regulation of B-Type Cyclin Proteolysis by Cdc28-Associated Kinases in Budding Yeast The EMBO Journal Vol.16 No.10 pp.2693–2702, 1997 Regulation of B-type cyclin proteolysis by Cdc28-associated kinases in budding yeast Angelika Amon cyclins are stable during S phase and mitosis, but are abruptly degraded at the end of mitosis (Evans et al., Whitehead Institute for Biomedical Research, 9 Cambridge Center, 1983). A sequence at the N-terminus of cyclin B, the Cambridge, MA 02142, USA destruction box, is required for this rapid degradation, and deletion of this nine amino acid motif stabilizes the protein In budding yeast, stability of the mitotic B-type cyclin (Glotzer et al., 1991). Overexpression of such a stabilized Clb2 is tightly cell cycle-regulated. B-type cyclin proteo- cyclin B causes arrest of the cell cycle in late anaphase lysis is initiated during anaphase and persists through- in a wide variety of organisms, including budding yeast out the G phase. Cln-Cdc28 kinase activity at START 1 (Murray et al., 1989; Ghiara et al., 1991; Luca et al., is required to repress B-type cyclin-specific proteolysis. 1991; Gallant and Nigg, 1992; Surana et al., 1993; Sigrist Here, we show that Clb-dependent kinases, when et al., 1995; Yamano et al., 1996). expressed during G , are also capable of repressing the 1 Degradation of cyclins is mediated by ubiquitin-depend- B-type cyclin proteolysis machinery. Furthermore, we ent proteolysis. Recently the components of this ubiquitin- find that inactivation of Cln- and Clb-Cdc28 kinases is sufficient to trigger Clb2 proteolysis and sister- conjugating machinery have been identified (reviewed by Deshaies, 1995; Glotzer, 1995). Attachment of ubiquitin chromatid separation in G2/M phase-arrested cells, where the B-type cyclin-specific proteolysis machinery to cyclin B is mediated by the ubiquitin-conjugating is normally inactive. Our results suggest that Cln- and enzymes UBC4 and UBCx (King et al., 1995; Aristarkhov Clb-dependent kinases are both capable of repressing et al., 1996; Yu et al., 1996) and a 20S ubiquitin ligase B-type cyclin-specific proteolysis and that they are complex known as the cyclosome or anaphase-promoting required to maintain the proteolysis machinery in an complex (APC; King et al., 1995; Sudakin et al., 1995; Zachariae et al., 1996). This complex is comprised of inactive state in S and G2/M phase-arrested cells. We propose that in yeast, as cells pass through START, eight subunits, four of which encode the previously Cln-Cdc28-dependent kinases inactivate B-type cyclin identified proteins Cdc16, Cdc23, Cdc27 and Apc1/BimE proteolysis. As Cln-Cdc28-dependent kinases decline which are highly conserved among eukaryotes (Irniger et al., 1995; King et al., 1995; Tugendreich et al., 1995; during G2, Clb-Cdc28-dependent kinases take over this role, ensuring that B-type cyclin proteolysis is not Peters et al., 1996; Zachariae et al., 1996). In yeast, activated during S phase and early mitosis. temperature-sensitive cdc16, cdc27 and cdc23 mutants Keywords: Cdc28-dependent kinases/Clb2/cyclin B arrest in metaphase when shifted to the restrictive tempera- proteolysis/Saccharomyces cerevisiae/Sic1 ture. This phenotype is consistent with the finding that proteins other than cyclin must be proteolysed in order for sister chromatids to segregate at the metaphase– anaphase transition (Holloway et al., 1993). Introduction B-type cyclin proteolysis is itself cell cycle-regulated. Protein kinases whose activity is regulated by cyclins Work in embryonic extracts has shown that the cyclosome (cyclin-dependent kinases; Cdks) govern cell cycle pro- or APC is the target for this cell cycle regulation (King gression. In the budding yeast Saccharomyces cerevisiae, et al., 1995; Sudakin et al., 1995). How cyclosome/ a single cyclin-dependent kinase, Cdc28, regulates passage APC activity is regulated during the cell cycle is poorly through the cell cycle. Its specificity is regulated by understood. In clams, the Cdc2/cyclin B-dependent kinases various cyclin proteins (reviewed by Nasmyth, 1993). are required for activation of B-type cyclin proteolysis Commitment to the cell cycle at START, spindle pole (Lahav-Baratz et al., 1995; Sudakin et al., 1995). However, body duplication and bud formation are triggered by active it is uncertain whether this effect is direct, because there kinase complexes composed of Cdc28 and the G1 cyclins is a lag phase between activation of Cdc2/cyclin B kinase Cln1, 2 and 3. Initiation of S phase requires Cdc28 and the activation of cyclin B degradation. In budding complexed with the S phase cyclins Clb5 and Clb6. yeast, B-type cyclin proteolysis is initiated during anaphase Formation of a mitotic spindle, spindle elongation and (Irniger et al., 1995) and continues to be active throughout nuclear division all rely on kinase complexes containing G1 (Amon et al., 1994). The Cln-dependent kinases are the B-type cyclins Clb1 to Clb5 (Nasmyth, 1993). required to repress Clb2 proteolysis as cells enter the cell Activity of the different Cdc28-dependent kinase com- cycle at START (Amon et al., 1994; Dirick et al., 1995). plexes is confined to appropriate stages of the cell cycle. The mechanism(s) by which Cln-dependent kinases repress Transcriptional control of cyclins and post-translational Clb2 proteolysis, how repression is maintained as the Cln- mechanisms restrict specific cyclin-dependent kinases to associated kinases decline during late S phase/G2, and certain stages of the cell cycle (reviewed by Koch and how Clb2 proteolysis is activated during mitosis is not Nasmyth, 1994; Nurse, 1994). B-type cyclin-specific understood. proteolysis is a key post-translational control. B-type Here, we investigate the role of Cln- and Clb-dependent © Oxford University Press 2693 A.Amon kinases in repression of B-type cyclin-specific proteolysis Amon et al., 1994). We therefore cannot exclude the during late G1, S phase and early mitosis. We find that possibility that cyclins other than Clb2, i.e. Clb5 and Clb-dependent kinases, like Cln-dependent kinases, can Clb6, contribute to the inactivation of B-type cyclin repress Clb2 proteolysis during G1. Furthermore, we show proteolysis. These results, however, show that inactivation that inactivation of Cln- and Clb-associated kinases is of Clb2 proteolysis is not specific to Cln-dependent sufficient to induce proteolysis of Clb2 and to trigger kinases, but that Clb-dependent kinases are also capable sister-chromatid segregation in S and G2/M phase-arrested of inactivating B-type cyclin proteolysis. cells where B-type cyclin proteolysis is normally inactive. These results suggest that Cln- and Clb-dependent kinases Inactivation of Clb- and Cln-dependent kinases are required to keep the B-type cyclin-specific proteolysis induces Clb2 decay in nocodazole-arrested cells machinery in an inactive state in S and G2/M phase- The requirement of Cdc28 kinase activity to switch off arrested cells. We propose that, as Cln-dependent kinase B-type cyclin proteolysis during G1 could be transient; levels decrease during G2, Clb-dependent kinases assume they could act only to initiate repression. Alternatively, the role of keeping B-type cyclin proteolysis inactive. Cln- and Clb-dependent kinases could be continuously required to maintain inhibition of B-type cyclin proteolysis Results throughout S phase, G2 and early mitosis. To address this question we analyzed the consequences of inactivating Clb cyclins can inactivate Clb2-specific proteolysis Clb- and Cln-dependent kinases on Clb2 stability in G2/M during G1 phase-arrested cells. The most direct way to address this Inactivation of Clb2 proteolysis at START requires Cln- question is to use a temperature-sensitive CDC28 allele. dependent kinase activity (Amon et al., 1994; Dirick et al., However, we have found that in the temperature-sensitive 1995), indicating that these kinases directly or indirectly alleles available, kinase complexes formed at the permis- repress B-type cyclin proteolysis. To further our under- sive temperature cannot be readily inactivated upon shift standing of how repression of B-type cyclin proteolysis to the restrictive temperature (A.Amon, unpublished is regulated, we determined whether inactivation of B-type observations). To inactivate Cln- and Clb-dependent cyclin proteolysis at START can be brought about solely by kinases efficiently, we took advantage of the fact that Cln-dependent kinases or whether Clb-dependent kinases, the cyclin-dependent kinase inhibitor (CDK inhibitor) when expressed during G1, are also capable of triggering Sic1 specifically inactivates Clb-dependent kinases inactivation of Clb2 proteolysis. (Mendenhall, 1993; Schwob et al., 1994) and that α-factor To this end we generated a strain which was deleted pheromone induces production of the CDK inhibitor for cln1, cln2 and cln3 but kept alive by expression of Far1, which specifically inhibits the Cln-dependent kinases CLN2 from the methionine-repressible MET3 promoter (reviewed by Cross, 1995). To inhibit Clb-dependent (Amon et al., 1994). In the presence of methionine, cells kinases efficiently a strain was constructed that contains arrest in G1 due to lack of G1 (Cln) cyclins. In addition, five copies of the SIC1 gene under the control of the this strain carried a single copy of either the wild-type galactose-inducible GAL1-10 promoter (GAL–SIC1 CLB2 gene (GAL–CLB2) or a version of CLB2 which strain). In the presence of galactose, cells arrest with a lacks the destruction box (GAL–CLB2-db∆) under the phenotype similar to that exhibited by cells lacking all control of the galactose-inducible GAL1-10 promoter. B-type cyclin-associated kinases (data not shown; Schwob During G1, Clb2 protein expressed from the GAL1-10 et al., 1994), indicating that Sic1 levels sufficient to inhibit promoter fails to accumulate due to continuous B-type all Clb-dependent kinases are generated. cyclin-specific proteolysis activity, whereas Clb2-db∆ pro- To analyze the consequences of inactivating Clb- and tein accumulates and forms active Clb2-associated kinase Cln-dependent kinases on Clb2 proteolysis, GAL–SIC1 during G1 (Amon et al., 1994).
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