Abstract
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.
Highlights
The ubiquitin-mediated proteolytic pathway has been implicated in multiple cellular processes, including endocytosis, cell cycle regulation, transcriptional activation, and antigen presentation
The ubiquitin ligase Itch belongs to the Nedd4/Rsp5p HECT domain-containing family of E3 ligases characterized by a number of regions that are involved in intracellular localization and protein-protein interactions, which include an N-terminal C2 domain, four WW domains, and a C-terminal HECT domain (Fig. 1A)
We have demonstrated that Endophilin A1, an Src homology 3 (SH3) domain-containing protein that functions in clathrin-mediated endocytosis is a substrate for Itch
Summary
The ubiquitin-mediated proteolytic pathway has been implicated in multiple cellular processes, including endocytosis, cell cycle regulation, transcriptional activation, and antigen presentation. HEK-293T cells were selected because they express detectable levels of both Itch and FAM/USP9X (Fig. 6B). Immunoprecipitation of endogenous Endophilin resulted in the coimmunoprecipitation of both FAM/USP9X and Itch (Fig. 2A).
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