Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation
Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation
复制标题
DOI：
10.1101/gad.13.9.1181
复制
发表时间：
1999-05-01
影响因子：
10.5
通讯作者：
Pagano, M
中科院分区：
文献类型：
--
作者：
Montagnoli, A;Fiore, F;Pagano, M
细胞周期蛋白依赖性激酶（Cdk）抑制剂p27的细胞丰度由泛素-蛋白酶体系统调节。p27降解的激活见于增殖细胞和许多类型的侵袭性人类癌。p27可以被Cdks磷酸化在苏氨酸187上，并且细胞周期蛋白E/Cdk 2过表达可以刺激野生型p27的降解，但不能刺激苏氨酸187-丙氨酸p27突变体[p27（T187 A）]的降解。然而，目前尚不清楚苏氨酸187磷酸化是否通过遍在蛋白-蛋白酶体系统或替代途径刺激p27降解。在这里，我们表明，p27泛素化（在体内和体外重组系统中测定）是细胞周期调节和Cdk活性所需的p27在体外泛素化。此外，野生型p27的泛素化，而不是p27（T187 A），可以发生在G（1）富集的提取物，只有在加入细胞周期蛋白E/Cdk 2或细胞周期蛋白A/Cdk 2。使用p27的磷酸苏氨酸187位点特异性抗体，我们发现p27的苏氨酸187磷酸化也是细胞周期依赖性的，存在于增殖细胞中，但在G（1）细胞中检测不到。最后，我们表明，除了苏氨酸187磷酸化，有效的p27泛素化需要形成一个三聚体复合物与细胞周期蛋白和Cdk亚基。事实上，cyclin B/Cdk 1能有效磷酸化p27，但不能与p27形成稳定的复合物，不能刺激G（1）提取物对p27的泛素化。此外，另一种p27突变体[p27（CK-）]可以被细胞周期蛋白E/Cdk 2磷酸化，但不能结合这种激酶复合物，对泛素化是难治的。因此，在整个细胞周期中，磷酸化和三聚体复合物的形成都充当Cdk抑制剂泛素化的信号。
The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G(1)-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G(1) cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G(1) extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor.