Purification of Hsk1, a minichromosome maintenance protein kinase from fission yeast
Purification of Hsk1, a minichromosome maintenance protein kinase from fission yeast
复制标题
DOI：
10.1074/jbc.273.34.22083
复制
发表时间：
1998-08-21
影响因子：
4.8
通讯作者：
Kelly, TJ
中科院分区：
文献类型：
--
作者：
Brown, GW;Kelly, TJ
Cdc 7蛋白激酶家族的成员对于所有真核生物中DNA复制的起始是必不可少的，但其精确的生物化学功能尚不清楚。我们已经将分裂酵母Cdc 7同源物Hsk 1纯化了大约30，000倍，接近同质。纯化的Hsk 1在几种底物上具有蛋白激酶活性，并且能够自磷酸化。Hsk 1高度保守区域的点突变在体外和体内破坏激酶。两个突变的Hsk 1等位基因的过量产生阻断DNA复制的起始并扰乱有丝分裂检查点，这与Hsk 1在起始早期阶段的作用一致。纯化的Hsk 1激酶可以被分离成两种活性形式，Hsk 1单体和由Hsk 1与共纯化多肽Dfp 1复合组成的异源二聚体。与Dfp 1的协会刺激外源性底物的磷酸化，但对自身激酶活性的影响不大。我们已经确定Dfp 1作为芽殖酵母Dbf 4的裂殖酵母同源物。纯化的Hsk 1磷酸化从裂殖酵母中纯化的六元微染色体维持蛋白复合物的Cdc 19（Mcm 2）亚基。由于微染色体维持蛋白与DNA复制的起始有关，Hsk 1在G（1）/S转换中的基本功能可能是由Cdc 19的磷酸化介导的。此外，Hsk 1激酶对关键底物的磷酸化可能通过与Dbf 4样辅因子的结合来调节。
Members of the Cdc7 family of protein kinases are essential for the initiation of DNA replication in all eukaryotes, but their precise biochemical function is unclear. We have purified the fission yeast Cdc7 homologue Hsk1 approximately 30,000-fold, to near homogeneity. Purified Hsk1 has protein kinase activity on several substrates and is capable of autophosphorylation, Point mutations in highly conserved regions of Hsk1 inactivate the kinase in vitro and in vivo, Overproduction of two of the mutant hsk1 alleles blocks initiation of DNA replication and deranges the mitotic checkpoint, a phenotype consistent with a role for Hsk1 in the early stages of initiation. The purified Hsk1 kinase can be separated into two active forms, a Hsk1 monomer and a heterodimer consisting of Hsk1 complexed with a copurifying polypeptide, Dfp1. Association with Dfp1 stimulates phosphorylation of exogenous substrates but has little effect on autokinase activity. We have identified Dfp1 as the fission yeast homologue of budding yeast Dbf4. Purified Hsk1 phosphorylates the Cdc19 (Mcm2) subunit of the six-member minichromosome maintenance protein complex purified from fission yeast, Since minichromosome maintenance proteins have been implicated in the initiation of DNA replication, the essential function of Hsk1 at the G(1)/S transition may be mediated by phosphorylation of Cdc19. Furthermore, the phosphorylation of critical substrates by Hsk1 kinase is likely regulated by association with a Dbf4-like co-factor.