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.
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.
Mouchantaf
Rania
R
Départment de sciences biologiques, Université de Montréal, Station Centre-Ville, Montreal, Quebec H3C 3J7, Canada.
Azakir
Bilal A
BA
McPherson
Peter S
PS
Millard
Susan M
SM
Wood
Stephen A
SA
Angers
Annie
A
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D006868:Hydrolysis; D015778:Minor Histocompatibility Antigens; D012097:Repressor Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D017931:DNA Primers; D015778:Minor Histocompatibility Antigens; D012097:Repressor Proteins; C481724:USP9Y protein, human; D025801:Ubiquitin; C432966:ITCH protein, human; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase