Burton J L, Solomon M J
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Mol Cell Biol. 2000 Jul;20(13):4614-25. doi: 10.1128/MCB.20.13.4614-4625.2000.
Ubiquitination and subsequent degradation of critical cell cycle regulators is a key mechanism exploited by the cell to ensure an irreversible progression of cell cycle events. The anaphase-promoting complex (APC) is a ubiquitin ligase that targets proteins for degradation by the 26S proteasome. Here we identify the Hsl1p protein kinase as an APC substrate that interacts with Cdc20p and Cdh1p, proteins that mediate APC ubiquitination of protein substrates. Hsl1p is absent in G(1), accumulates as cells begin to bud, and disappears in late mitosis. Hsl1p is stabilized by mutations in CDH1 and CDC23, both of which result in compromised APC activity. Unlike Hsl1p, Gin4p and Kcc4p, protein kinases that have sequence homology to Hsl1p, were stable in G(1)-arrested cells containing active APC. Mutation of a destruction box motif within Hsl1p (Hsl1p(db-mut)) stabilized Hsl1p. Interestingly, this mutation also disrupted the Hsl1p-Cdc20p interaction and reduced the association between Hsl1p and Cdh1p in coimmunoprecipitation studies. These findings suggest that the destruction box motif is required for Cdc20p and, to a lesser extent, for Cdh1p to target Hsl1p to the APC for ubiquitination. Hsl1p has been previously shown to inhibit Swe1p, a protein kinase that negatively regulates the cyclin-dependent kinase Cdc28p, by promoting Swe1p degradation via SCF(Met30) in a bud morphogenesis checkpoint. Results of the present work indicate that Hsl1p is degraded in an APC-dependent manner and suggest a link between the SCF (Skp1-cullin-F box) and APC-proteolytic systems that may help to coordinate the proper progression of cell cycle events.
关键细胞周期调节因子的泛素化及随后的降解是细胞用来确保细胞周期事件不可逆进展的关键机制。后期促进复合物（APC）是一种泛素连接酶，它将蛋白质靶向26S蛋白酶体进行降解。在这里，我们确定Hsl1p蛋白激酶是一种APC底物，它与Cdc20p和Cdh1p相互作用，Cdc20p和Cdh1p是介导APC对蛋白质底物进行泛素化的蛋白质。Hsl1p在G1期不存在，随着细胞开始出芽而积累，并在有丝分裂后期消失。Hsl1p通过CDH1和CDC23中的突变而稳定，这两者都会导致APC活性受损。与Hsl1p不同，与Hsl1p具有序列同源性的蛋白激酶Gin4p和Kcc4p在含有活性APC的G1期停滞细胞中是稳定的。Hsl1p中一个破坏框基序的突变（Hsl1p(db-mut)）使Hsl1p稳定。有趣的是，这个突变也破坏了Hsl1p与Cdc20p的相互作用，并在共免疫沉淀研究中减少了Hsl1p与Cdh1p之间的关联。这些发现表明，破坏框基序是Cdc20p将Hsl1p靶向APC进行泛素化所必需的，在较小程度上也是Cdh1p所必需的。先前已表明，Hsl1p通过在芽形态发生检查点中经由SCF(Met30)促进Swe1p降解来抑制Swe1p，Swe1p是一种负调节细胞周期蛋白依赖性激酶Cdc28p的蛋白激酶。目前的工作结果表明，Hsl1p以APC依赖的方式被降解，并提示SCF（Skp1-遍在蛋白连接酶E3的一个亚基-F盒蛋白）和APC蛋白水解系统之间存在联系，这可能有助于协调细胞周期事件的正常进展。
