Activating phosphorylation of the Saccharomyces cerevisiae cyclin-dependent kinase, cdc28p, precedes cyclin binding.
Activating phosphorylation of the Saccharomyces cerevisiae cyclin-dependent kinase, cdc28p, precedes cyclin binding.
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DOI：
10.1091/mbc.11.5.1597
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发表时间：
2000-05
影响因子：
3.3
通讯作者：
K. Ross;P. Kaldis;M. Solomon
中科院分区：
文献类型：
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作者：
K. Ross;P. Kaldis;M. Solomon
真核细胞周期进程由称为细胞周期蛋白依赖性激酶（Cdks）的蛋白激酶家族控制。Cdk的激活有两个步骤：细胞周期蛋白的结合和Cdk激活激酶（CAK）对保守的苏氨酸残基的磷酸化。我们已经研究了这些监管机制之间的相互作用，在激活的主要酿酒酵母Cdk，Cdc 28 p。我们发现，大多数Cdc 28 p磷酸化的激活苏氨酸（Thr-169）在整个细胞周期。Thr-169磷酸化的程度是类似的单体Cdc 28 p和Cdc 28 p结合细胞周期蛋白。通过改变加入细胞周期蛋白和Cak 1 p的顺序，我们确定Cdc 28 p在细胞周期蛋白结合之前磷酸化时被最有效地激活。此外，我们发现，Cdc 28 p（T169 A）突变体，不能磷酸化，结合细胞周期蛋白不如野生型Cdc 28 p在体内。这些结果表明，未磷酸化的Cdc 28 p可能无法紧密结合细胞周期蛋白。我们建议，Cdc 28 p通常是磷酸化的Cak 1 p之前，它结合细胞周期蛋白。这种激活途径与高等真核生物中的细胞周期蛋白结合似乎先于激活磷酸化形成对比。
Eukaryotic cell cycle progression is controlled by a family of protein kinases known as cyclin-dependent kinases (Cdks). Two steps are essential for Cdk activation: binding of a cyclin and phosphorylation on a conserved threonine residue by the Cdk-activating kinase (CAK). We have studied the interplay between these regulatory mechanisms during the activation of the major Saccharomyces cerevisiae Cdk, Cdc28p. We found that the majority of Cdc28p was phosphorylated on its activating threonine (Thr-169) throughout the cell cycle. The extent of Thr-169 phosphorylation was similar for monomeric Cdc28p and Cdc28p bound to cyclin. By varying the order of the addition of cyclin and Cak1p, we determined that Cdc28p was activated most efficiently when it was phosphorylated before cyclin binding. Furthermore, we found that a Cdc28p(T169A) mutant, which cannot be phosphorylated, bound cyclin less well than wild-type Cdc28p in vivo. These results suggest that unphosphorylated Cdc28p may be unable to bind tightly to cyclin. We propose that Cdc28p is normally phosphorylated by Cak1p before it binds cyclin. This activation pathway contrasts with that in higher eukaryotes, in which cyclin binding appears to precede activating phosphorylation.