Cell cycle-dependent phosphorylation of human CDC5 regulates RNA processing
Cell cycle-dependent phosphorylation of human CDC5 regulates RNA processing
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DOI：
10.4161/cc.7.12.6017
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发表时间：
2008-06-15
期刊：
影响因子：
4.3
通讯作者：
Bernstein, Harold S.
中科院分区：
文献类型：
--
作者：
Graub, Remo;Lancero, Hope;Bernstein, Harold S.
CDC5蛋白是前mrna剪接复合体的组成部分，对酵母、植物和哺乳动物的细胞周期进程至关重要。人类CDC5以有丝分裂原依赖的方式磷酸化，其与剪接体的关联是atp依赖的。对氨基酸序列的检查表明，CDC5L可能在多达28个已知激酶的潜在共识识别序列上被磷酸化，然而，实际磷酸化位点的身份，它们在调节CDC5L活性中的作用，以及负责它们磷酸化的激酶之前尚未确定。通过二维磷酸肽定位和纳米电喷雾质谱分析，我们发现CDC5L在体内至少有9个位点被磷酸化。我们证明，虽然CDC5L能够在体外和体内形成同型二聚体，但同型二聚体化和核定位都不依赖于这些位点的磷酸化。通过体外剪接实验，我们发现CDC5L介导的pre-mRNA剪接需要CDC5L在CDKs识别序列中苏氨酸411和438位点的磷酸化。我们还证明了CDK2的特异性抑制剂CVT-313在体外激酶测定和体内循环细胞的放射性标记实验中抑制CDC5L的磷酸化。这些研究首次证明了CDC5L磷酸化的调节作用，并表明靶向这些位点或相关激酶可能为治疗无保护的细胞增殖疾病（如癌症）提供新的策略。
CDC5 proteins are components of the pre-mRNA splicing complex and essential for cell cycle progression in yeast, plants and mammals. Human CDC5 is phosphorylated in a mitogen-dependent manner, and its association with the spliceosome is ATP-dependent. Examination of the amino acid sequence suggests that CDC5L may be phosphorylated at up to 28 potential consensus recognition sequences for known kinases, however, the identity of actual phosphorylation sites, their role in regulating CDC5L activity, and the kinases responsible for their phosphorylation have not previously been determined. Using two-dimensional phosphopeptide mapping and nanoelectrospray mass spectrometry, we now show that CDC5L is phosphorylated on at least nine sites in vivo. We demonstrate that while CDC5L is capable of forming homodimers in vitro and in vivo, neither homodimerization nor nuclear localization is dependent on phosphorylation at these sites. Using an in vitro splicing assay, we show that phosphorylation of CDC5L at threonines 411 and 438 within recognition sequences for CDKs are required for CDC5L-mediated pre-mRNA splicing. We also demonstrate that a specific inhibitor of CDK2, CVT-313, inhibits CDC5L phosphorylation in both in vitro kinase assays and in vivo radiolabeling experiments in cycling cells. These studies represent the first demonstration of a regulatory role for phosphorylation of CDC5L, and suggest that targeting these sites or the implicated kinases may provide novel strategies for treating disorders of unguarded cellular proliferation, such as cancer.