Analysis of the CK2-dependent phosphorylation of serine 13 in Cdc37 using a phospho-specific antibody and phospho-affinity gel electrophoresis
Analysis of the CK2-dependent phosphorylation of serine 13 in Cdc37 using a phospho-specific antibody and phospho-affinity gel electrophoresis
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DOI：
10.1111/j.1742-4658.2007.06090.x
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发表时间：
2007-11-01
期刊：
影响因子：
5.4
通讯作者：
Nishida, Eisuke
中科院分区：
文献类型：
--
作者：
Miyata, Yoshihiko;Nishida, Eisuke
细胞分裂周期蛋白37(CDC37)中Ser13的磷酸化依赖于CK2，CDC37是一种激酶特异性的热休克蛋白90(HSP90)辅伴侣蛋白，此前已被报道在CDC37与信号蛋白激酶的联系中起关键作用[Bandhakavi S，McCann RO，Hanna DE&Glover CVC(2003)J Biol Chem278,2829-2836；Shao J，Prince T，Hartson SD&Matts RL(2003)J Biol Chem278,38117-38220；Miyata Y&Nishida E(2004)Mol Cell Biol24,4065-4074]。在这里，我们描述了一种新的针对CDC37的磷酸化特异性抗体，该抗体只有在有镁离子和ATP存在的情况下与CK2孵育时才能识别重组纯化的CDC37。CDC37中的Ser13被不可磷酸化的氨基酸取代，取消了与该抗体的结合。该抗体对磷酸化的CDC37具有特异性，与其他CK2底物如Hsp90和FK506结合蛋白52不发生交叉反应。利用该抗体，我们在体内证明了Hsp90与其客户信号通路CDK4、MOK、v-Src和Raf1的复合体中含有CK2磷酸化形式的CDc37。免疫荧光染色显示，Hsp90和磷酸化形式的CDC37在表皮生长因子诱导的膜皱纹中聚集。我们进一步用磷酸化亲和凝胶电泳法鉴定了CDC37的磷酸化。我们的分析表明，依赖于CK2的Ser13上的CDC37的磷酸化导致了特定的凝胶迁移率的变化，并且Hsp90与其客户信号蛋白激酶之间的复合体中的CDC37以磷酸化的形式存在。我们的结果显示了CK2依赖的CDC37磷酸化的生理重要性，以及磷酸化亲和凝胶电泳法在蛋白质磷酸化分析中的应用。
The CK2-dependent phosphorylation of Ser13 in cell division cycle protein 37 (Cdc37), a kinase-specific heat shock protein 90 (Hsp90) cochaperone, has previously been reported to be essential for the association of Cdc37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem278, 2829-2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem278, 38117-38220; Miyata Y & Nishida E (2004) Mol Cell Biol24, 4065-4074]. Here we describe a new phospho-specific antibody against Cdc37 that recognizes recombinant purified Cdc37 only when incubated with CK2 in the presence of Mg2+ and ATP. The replacement of Ser13 in Cdc37 by nonphosphorylatable amino acids abolished binding to this antibody. The antibody was specific for phosphorylated Cdc37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506-binding protein 52. Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v-Src, and Raf1, contained the CK2-phosphorylated form of Cdc37 in vivo. Immunofluorescent staining showed that Hsp90 and the phosphorylated form of Cdc37 accumulated in epidermal growth factor-induced membrane ruffles. We further characterized the phosphorylation of Cdc37 using phospho-affinity gel electrophoresis. Our analyses demonstrated that the CK2-dependent phosphorylation of Cdc37 on Ser13 caused a specific gel mobility shift, and that Cdc37 in the complexes between Hsp90 and its client signaling protein kinases was in the phosphorylated form. Our results show the physiological importance of CK2-dependent Cdc37 phosphorylation and the usefulness of phospho-affinity gel electrophoresis in protein phosphorylation analysis.