Cds1 Phosphorylation by Rad3-Rad26 Kinase Is Mediated by Forkhead-associated Domain Interaction with Mrc1*
Cds1 Phosphorylation by Rad3-Rad26 Kinase Is Mediated by Forkhead-associated Domain Interaction with Mrc1*
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DOI：
10.1074/jbc.m404834200
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发表时间：
2004-07
影响因子：
4.8
通讯作者：
Katsunori Tanaka;P. Russell
中科院分区：
文献类型：
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作者：
Katsunori Tanaka;P. Russell
蛋白激酶CDS1是裂殖酵母Pombe中复制检查点的效应者。CDS1是稳定停滞的复制分叉所必需的，它有助于防止有丝分裂的开始，直到基因组完全复制。MRc1(复制检查点-1的介体)和Rad3-Rad26激酶是CDS1激活所必需的，但目前还不清楚MRc1是如何介导CDS1的激活的。在这里，我们证明了MRc1是RAD3-RAD26初始苏氨酸11磷酸化CDS1所必需的。在酵母双杂交试验中，MRc1与CDs1的叉头相关(FHA)结构域特异地相互作用。FHA结构域的突变取消了这种相互作用，也消除了CDS1的Thr-11磷酸化。野生型CDs1的共表达挽救了CDs1“激酶死亡”突变体的微弱的Thr-11磷酸化。RAD26-CDS1融合蛋白的表达可以部分消除复制检查点对MRc1的需求。这些发现表明，CDS1的FHA结构域对MRc1的识别有助于将CDS1招募到Rad3-Rad26。这种相互作用介导了RAD3-RAD26对CDS1的初始Thr-11磷酸化，以及随后的分子间磷酸化事件，导致CDS1的完全激活。
The protein kinase Cds1 is an effector of the replication checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required to stabilize stalled replication forks, and it helps to prevent the onset of mitosis until the genome is fully replicated. Mrc1 (mediator of the replication checkpoint-1) and Rad3-Rad26 kinase are required for Cds1 activation, but exactly how Mrc1 mediates Cds1 activation is unknown. Here we show that Mrc1 is required for the initial threonine 11 phosphorylation of Cds1 by Rad3-Rad26. Mrc1 specifically interacts with the forkhead-associated (FHA) domain of Cds1 in yeast two-hybrid assays. Mutations in the FHA domain that abolish this interaction also eliminate Thr-11 phosphorylation of Cds1. Weak Thr-11 phosphorylation of a “kinase-dead” mutant of Cds1 is rescued by co-expression of wild type Cds1. The requirement for Mrc1 in the replication checkpoint can be partially eliminated by expression of a Rad26-Cds1 fusion protein. These findings suggest that recognition of Mrc1 by the FHA domain of Cds1 serves to recruit Cds1 to Rad3-Rad26. This interaction mediates the initial Thr-11 phosphorylation of Cds1 by Rad3-Rad26 with subsequent intermolecular phosphorylation events leading to full activation of Cds1.