The ubiquitin ligase itch is auto-ubiquitylated in vivo and in vitro but is protected from degradation by interacting with the deubiquitylating enzyme FAM/USP9X
The ubiquitin ligase itch is auto-ubiquitylated in vivo and in vitro but is protected from degradation by interacting with the deubiquitylating enzyme FAM/USP9X
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DOI：
10.1074/jbc.m605959200
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发表时间：
2006-12-15
影响因子：
4.8
通讯作者：
Angers, Annie
中科院分区：
文献类型：
--
作者：
Mouchantaf, Rania;Azakir, Bilal A.;Angers, Annie
瘙痒是一种泛素连接酶，参与了许多细胞过程的调节。我们以前已经鉴定出Itch是内吞蛋白Endothinin的结合伙伴，并发现它定位于内小体。利用亲和纯化与质谱联用的方法，我们现在已经确定泛素蛋白水解酶FAM/USP9X是Itch的结合伙伴。体外用谷胱甘肽S转移酶下拉试验和体内免疫共沉淀法证实了Ich与FAM/USP9X的相关性。瘙痒和FAM部分共存于COS-7细胞的跨高尔基网处和外周小泡中。我们将Itch上的FAM结合结构域映射到WW结构域，这是一个已知参与底物识别的区域。然而，FAM/USP9X的瞬时过表达导致了Itch的去泛素化。此外，我们还证明了Itch自身泛素化导致其在蛋白酶体中的降解。通过检测不同细胞系和大鼠组织中Ich和FAM的含量，发现这两种蛋白的表达呈正相关。这一观察表明，FAM的表达水平可能对细胞内的瘙痒有影响。通过RNA干扰实验降低FAM水平导致细胞内内源性Ich水平显著降低，这可以通过蛋白酶体抑制剂lactacystin的治疗来预防。相应地，FAM/USP9X的过表达导致内源性瘙痒水平显著增加。这些结果表明，基于两种蛋白质之间的直接相互作用，泛素连接酶和泛素蛋白水解酶之间存在着有趣的相互作用。
Itch is a ubiquitin ligase that has been implicated in the regulation of a number of cellular processes. We previously have identified Itch as a binding partner for the endocytic protein Endophilin and found it to be localized to endosomes. Using affinity purification coupled to mass spectrometry, we have now identified the ubiquitin-protease FAM/USP9X as a binding partner of Itch. The association between Itch and FAM/USP9X was confirmed in vitro by glutathione S-transferase pulldown and in vivo through coimmunoprecipation. Itch and FAM partially colocalize in COS-7 cells at the trans-Golgi network and in peripheral vesicles. We mapped the FAM-binding domain on Itch to the WW domains, a region known to be involved in substrate recognition. However, transient overexpression of FAM/USP9X resulted in the deubiquitylation of Itch. Moreover, we show that Itch auto-ubiquitylation leads to its degradation in the proteasome. By examining the amounts of Itch and FAM in various cell lines and rat tissues, a positive correlation was found in the expression of both proteins. This observation suggests that the levels of FAM expression could have an influence on Itch in cells. Experimental decrease in FAM levels by RNA interference leads to a significant reduction in intracellular levels of endogenous Itch, which can be prevented by treatment with the proteasome inhibitor lactacystin. Accordingly, overexpression of FAM/USP9X resulted in a marked increase in endogenous Itch levels. These results demonstrate an intriguing interplay between a ubiquitin ligase and a ubiquitin protease, based on direct interaction between the two proteins.