Control of AMPK-related kinases by USP9X and atypical Lys29/Lys33-inked polyubiquitin chains
Control of AMPK-related kinases by USP9X and atypical Lys29/Lys33-inked polyubiquitin chains
复制标题
DOI：
10.1042/bj20080067
复制
发表时间：
2008-04-15
影响因子：
4.1
通讯作者：
Alessi, Dario R.
中科院分区：
文献类型：
--
作者：
Al-Hakim, Abdallah K.;Zagorska, Anna;Alessi, Dario R.
AMPK（AMP活化蛋白激酶）相关激酶调节细胞极性以及增殖，并由LKB 1-肿瘤抑制激酶激活。在本研究中，我们证明了AMPK相关激酶NUAK 1（AMPK相关激酶5）和MARK 4（微管亲和力调节激酶4）在体内是多泛素化的，并与去泛素化酶USP 9 X（泛素特异性蛋白酶9）相互作用。USP 9 X的敲低增加NUAK 1和MARK 4的多聚泛素化，而USP 9 X的过表达抑制泛素化。USP 9 X催化从野生型NUAK 1中去除聚泛素链，但不从非USP 9 X结合突变体中去除。拓扑分析显示，连接到NUAK 1和MARK 4的泛蛋白单体是通过Lys（29）和/或Lys（33）连接的，而不是更常见的Lys（48）/Lys（63）连接的。我们发现AMPK和其他AMPK相关激酶在细胞中也是多泛素化的。我们鉴定了NUAK 1和MARK 4的非USP 9 X结合突变体，发现这些突变体在细胞中表达时在LKB 1靶向的T环残基处高度泛素化且不磷酸化，这表明多聚泛素化可能抑制这些酶。本研究的结果表明，NUAK 1和MARK 4是USP 9 X的底物，并提供了AMPK家族激酶受不寻常的Lys（29）/Lys（33）连接的多聚泛素链调节的第一个证据。
AMPK (AMP-activated protein kinase)-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. In the present study we demonstrate that the AMPK-related kinases, NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4), are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X (ubiquitin specific protease-9). Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whereas overexpression of USP9X inhibited ubiquitination. USP9X, catalysed the removal of polyubiquitin chains from wild-type NUAK1, but not from a non-USP9X-binding mutant. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys(29) and/or Lys(33) rather than the more common Lys(48)/Lys(63). We find that AMPK and other AMPK-related kinases are also polyubiquitinated in cells. We identified non-USP9X-binding mutants of NUAK1 and MARK4 and find that these are hyper-ubiquitinated and not phosphorylated at their T-loop residue targeted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. The results of the present study demonstrate that NUAK1 and MARK4 are substrates of USP9X and provide the first evidence that AMPK family kinases are regulated by unusual Lys(29)/Lys(33)-linked polyubiquitin chains.