Regulation of Ubiquitin-like with PHD and RING Finger Domain 1 ( UHRF 1 ) Protein Stability by Heat Shock Protein 90 Chaperone Machinery

As a protein critical for DNA maintenance methylation and cell proliferation, UHRF1 is frequently highly expressed in various human cancers and is considered as a drug target for cancer therapy. In a high throughput screening for small molecules that induce UHRF1 protein degradation, we have identified the Hsp90 inhibitor 17allylamino-17-demethoxygeldanamycin (17-AAG). We present evidence that UHRF1 interacts with Hsp90 chaperone complex and is a novel Hsp90 client protein. Pharmacological inhibition of Hsp90 with 17-AAG or 17-DMAG results in UHRF1 ubiquitination and proteasome-dependent degradation. Interestingly this Hsp90 inhibitorinduced UHRF1 degradation is independent of CHIP and CUL5, two previously identified ubiquitin E3 ligases for Hsp90 client proteins. In addition, this degradation is dependent neither on the UHRF1’s intrinsic E3 ligase nor the E3 ligase SCF that has been implicated in regulation of UHRF1 stability. We also http://www.jbc. org/cgi/doi/10.1074/jbc.M116.727214 The latest version is at JBC Papers in Press. Published on August 3, 2016 as Manuscript M116.727214 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 6, 2017 hp://w w w . jb.org/ D ow nladed from Hsp90 inhibitors induce UHRF1 degradation 2 provide evidence that Hsp90 inhibitors may suppress cancer cell proliferation in part through its induced UHRF1 degradation. Taken together, our results identify UHRF1 as a novel Hsp90 client protein and shed light on the regulation of UHRF1 stability and function. Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) is originally isolated in a yeast-one-hybrid screen as a protein that binds to the CCAAT box within the topoisomerase II α gene promoter (1). UHRF1 (also known as ICBP90 in humans and Np95 in mice) is now well recognized for its essential role in DNA maintenance methylation via targeting DNMT1 to DNA replication forks during the S phase of cell cycle (2,3). UHRF1 itself binds to DNA replication forks through a coordinated recognition of hemimethylated CpG dinucleotides by its unique SRA domain and histone H3 tails with dior tri-methylated lysine 9 (H3K9me2/3) by a concerted function of a TTD (tandem Tudor domain) and PHD domain (4-9). DNMT1 is then recruited most likely through a physical interaction with both UHRF1 and ubiquitinated H3, the latter is catalyzed by the E3 ligase activity of UHRF1 (6,10-12). In addition to its critical role in DNA methylation, UHRF1 is also known as a key regulator of cell proliferation. Numerous studies have shown that UHRF1 is overexpressed in various cancers and its overexpression correlates with tumor progression (13-15). Moreover, several studies have demonstrated that overexpression of UHRF1 drives cell proliferation, whereas knockdown of UHRF1 results in a strong cell cycle arrest, hypersensitivity to DNA damage and chemotherapeutic agents and increased apoptosis (16-18). Collectively these studies have led to the proposal that UHRF1 is not only a tumor biomarker but also a potential target for cancer therapy. However currently there is no reported small molecule that either inhibits UHRF1 activity or induces its degradation. Hsp90 (90 kDa heat-shock protein) is a most abundant and conserved molecular chaperone essential in eukaryotic cells (19). Unlike Hsp70, Hsp90 is not required for de novo folding of most proteins but promotes the final maturation of a selected group of proteins termed as Hsp90 client proteins (20). The Hsp90 client proteins are enriched of protein kinases, transcription factors, nuclear steroid receptors and regulatory proteins (21,22). Most commonly, Hsp90 facilitates their stabilization and activation and inactivation of Hsp90 by small molecule inhibitors such as the 17allylamino-17-desmethoxygeldanamycin (17-AAG) often leads to their degradation through ubiquitin– proteasome system (20-22). Given that numerous oncoproteins have been shown to be Hsp90’s client proteins and that Hsp90 is highly expressed in tumors compared to normal tissues, the development of Hsp90 inhibitors has become a new strategy in cancer therapy (23). In an effort to screen for small molecules that affect UHRF1 stability, we identified the Hsp90 inhibitor 17AAG as a potent inducer of UHRF1 degradation. We present evidence that UHRF1 specifically interacts with Hsp90 and is a novel Hsp90 client protein. by gest on Sptem er 6, 2017 hp://w w w . jb.org/ D ow nladed from Hsp90 inhibitors induce UHRF1 degradation