HAUSP regulates c-MYC expression via de-ubiquitination of TRRAP.
OBJECTIVE
The de-ubiquitinase HAUSP has been reported to exhibit various biological roles implicated in the development of cancer and other pathologies. The dual nature of HAUSP (i.e., oncogenic and tumor suppressive) makes the protein even more versatile. The major aims of this study were to reveal the effect of HAUSP over-expression on the overall proteome and to identify bona fide substrates of HAUSP. In addition, we aimed to unravel the functionality and physiological relevance of the de-ubiquitinating activity of HAUSP on one of its newly identified substrates, TRRAP.


METHODS
An overall proteome analysis was performed after exogenous HAUSP over-expression in HEK293 cells, followed by 2-dimensional gel electrophoresis (2-DE). Interacting proteins were subsequently isolated using immunoprecipitation and 1-dimensional gel electrophoresis (1-DE). Both were followed by tandem MALDI-TOF/TOF mass spectrometry and gene ontology-based analyses. To validate the functionality of one of the identified substrates (TRRAP), Western blotting, immunocytochemistry, immunoprecipitation, in vivo de-ubiquitination, quantitative real-time PCR and luciferase assays were performed.


RESULTS
The substrate screening indicated that HAUSP may be involved in tumorigenesis, cytoskeletal organization and transport, and chaperone systems. One candidate substrate, TRRAP, was found to physically interact and co-localize with HAUSP. As TRRAP regulates c-MYC expression, and in order to validate the effect of HAUSP on TRRAP, c-MYC protein and mRNA expression levels were analyzed after exogenous HAUSP over-expression. Both were found to be up-regulated. We also found that c-MYC transactivation increased upon exogenous HAUSP over-expression. By using a luciferase reporter assay, we found that a c-MYC responsive promoter exhibited increased activity, which was subsequently abrogated upon TRRAP knockdown.


CONCLUSIONS
From our results we conclude that HAUSP may act as an oncogenic protein that can modulate c-MYC expression via TRRAP. Our results provide a new context in which HAUSP may play a role in cancer cell signalling.
Signal Transduction in Cancer and Stem Cells Laboratory, Division of Cancer Biology and Inflammatory Disorder, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata, 700 032, India.
Seemana
S
Bhattacharya
Mrinal K
MK
Ghosh
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D057809:HEK293 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*; D000076084:Ubiquitin-Specific Peptidase 7; D054875:Ubiquitination
D048868:Adaptor Proteins, Signal Transducing; D009687:Nuclear Proteins; D020543:Proteome; D016271:Proto-Oncogene Proteins c-myc; C113829:transformation-transcription domain-associated protein; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7