Wang Yang, Zhan Qimin
State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
J Biol Chem. 2007 Jun 15;282(24):17712-9. doi: 10.1074/jbc.M701350200. Epub 2007 Apr 2.
The recently identified centrosome protein Nlp (ninein-like protein) is a key regulator in centrosome maturation, which contributes to chromosome segregation and cytokinesis. However, the mechanism(s) controlling Nlp expression remains largely unknown. Here we have shown that Nlp expression is cell cycle-dependent with a peak at G(2)/M transition in human cells. Nlp is a short-lived protein and degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. It interacts with the APC/c through the APC2 or Cdc27 subunits and is ubiquitinated. Following treatment with proteasome inhibitors, its protein level is elevated. Nlp binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, and overexpression of Cdh1 and Cdc20 enhances Nlp degradation. Using point mutations of the two putative degradation signals in Nlp, we have found that its degradation requires intact KEN-box and D-box. Interestingly, the Lys-Glu-Asn-D-box-mutated Nlp exhibits a much stronger capability of inducing anchorage-independent growth and multinuclearity compared with the wild type Nlp. Taken together, these findings indicate that Nlp expression is cell cycle-dependent and regulated by APC-mediated protein degradation.
最近发现的中心体蛋白Nlp（九蛋白样蛋白）是中心体成熟过程中的关键调节因子，它有助于染色体分离和胞质分裂。然而，控制Nlp表达的机制在很大程度上仍不清楚。在这里，我们表明Nlp表达在人类细胞中依赖细胞周期，在G(2)/M转换期达到峰值。Nlp是一种半衰期短的蛋白质，通过后期促进复合体（APC/c）途径被蛋白酶体降解。它通过APC2或Cdc27亚基与APC/c相互作用并被泛素化。用蛋白酶体抑制剂处理后，其蛋白质水平升高。Nlp在体内与降解靶向蛋白Cdh1和Cdc20结合，Cdh1和Cdc20的过表达增强Nlp的降解。利用Nlp中两个假定降解信号的点突变，我们发现其降解需要完整的KEN盒和D盒。有趣的是，与野生型Nlp相比，赖氨酸-谷氨酸-天冬酰胺-D盒突变的Nlp表现出更强的诱导非锚定依赖性生长和多核化的能力。综上所述，这些发现表明Nlp表达依赖细胞周期，并受APC介导的蛋白质降解调控。
