Deubiquitination of Chfr, a checkpoint protein, by USP7/HAUSP regulates its stability and activity

Department of Biology, Kyung Hee University, Sŏul, Seoul, South Korea
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 07/2007; 357(3):615-9. DOI: 10.1016/j.bbrc.2007.03.193
Source: PubMed


Chfr, a mitotic stress checkpoint, plays an important role in cell cycle progression, tumor suppression and the processes that require the E3 ubiquitin ligase activity mediated by the RING finger domain. Chfr stimulates the formation of polyubiquitin chains by ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins including Plk1 and Aurora A. In this study, we identified USP7 (also known as HAUSP), which is a member of a family of proteins that cleave polyubiquitin chains and/or ubiquitin precursors, as an interacting protein with Chfr by immunoaffinity purification and mass spectrometry, and their interaction greatly increases the stability of Chfr. In fact, USP7 can remove ubiquitin moiety from the autoubiquitinated Chfr both in vivo and in vitro, which results in the accumulation of Chfr in the cell. Thus, our finding suggests that USP7-mediated deubiquitination of Chfr leads to its accumulation, which might be a key regulatory step for Chfr activation and that USP7 may play an important role in the regulation of Chfr-mediated cellular processes including cell cycle progression and tumor suppression.

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    • "USP7 has been described to protect the RING-finger E3-ubiquitin ligases ICP0 (49), Chfr (50) and Mdm2 (51) from autoubiquitinylation, thereby stabilizing their protein levels. We therefore transfected USP7 into H1299 cells to monitor the effect of USP7 on the stability of transfected and endogenous UHRF1 protein (Figure 3C and D). "
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    ABSTRACT: Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo.
    Full-text · Article · Jul 2011 · Nucleic Acids Research
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    ABSTRACT: Breast cancer can develop when genes that control the cell cycle and genomic stability are aberrantly expressed or non-functional. CHFR encodes an E3 ubiquitin ligase that reportedly delays mitosis in response to microtubule-targeting drugs (i.e. nocodazole and taxanes). Loss of CHFR mRNA expression has been reported in many cancers, including breast cancer, but the relevance of this to tumorigenesis remains unknown. To test if CHFR was relevant for mammary tumorigenesis, we analyzed the effects of altered expression in breast cancers. Nearly 40% of cultured and primary breast cancers had low or no CHFR, which was associated with large tumor size in patient samples. Decreased CHFR expression by RNAi in immortalized human mammary epithelial cell (IHMEC) lines resulted in taxane sensitivity and the acquisition of tumorigenic phenotypes including faster growth rates, higher mitotic indices, enhanced invasiveness and motility, epithelial-to-mesenchymal transitions, increased aneuploidy, and colony formation in soft agar. Conversely, over-expressing CHFR in breast cancer cells caused slower growth and decreased invasiveness and motility. To determine if CHFR was critical for genomic stability, cells transfected with CHFR siRNA were analyzed for chromosome segregation defects. Transient CHFR loss led to increased aneuploidy, misaligned metaphase chromosomes, anaphase bridges, multi-polar condensed spindles, multi-nucleated cells, and mislocalization of the mitotic checkpoint proteins MAD2 and BUBR1. CHFR was found to interact with three proteins required for mitotic spindle formation and chromosome segregation, including MAD2 and Aurora A where CHFR loss led to elevated Aurora A oncoprotein levels, but no change in MAD2 expression. Alpha-tubulin was identified as a novel target for CHFR-mediated ubiquitination and degradation after treatment with nocodazole. Decreased CHFR increased acetylated alpha-tubulin, a mitotic spindle protein implicated in cellular response to taxane treatment. These findings indicate that CHFR has tumor suppressive qualities and may be a biomarker for chemotherapeutic response to taxanes. CHFR has a previously unrecognized role as a regulator of genomic stability via its functional impact on BUBR1, MAD2, Aurora A, and alpha-tubulin. CHFR may be one of the few proteins that can control the cell cycle, chemotherapeutic response, and genomic stability - processes that go awry in breast cancer. Ph.D. Human Genetics University of Michigan, Horace H. Rackham School of Graduate Studies
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