Regulation of Ubiquitin-like with Plant Homeodomain and RING Finger Domain 1 (UHRF1) Protein Stability by Heat Shock Protein 90 Chaperone Machinery

Guangjin Ding,Peilin Chen,Hui Zhang,Xiaojie Huang,Yi Zang,Jiwen Li,Jia Li,Jiemin Wong

doi:10.1074/jbc.m116.727214

Abstract

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 17-allylamino-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-dimethylaminoethylamino-17-demethoxygeldanamycin results in UHRF1 ubiquitination and proteasome-dependent degradation. Interestingly, this HSP90 inhibitor-induced 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 intrinsic E3 ligase of UHRF1 nor on the E3 ligase SCF(β-TRCP) that has been implicated in regulation of UHRF1 stability. We also 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.

Highlights

Ubiquitin-like with PHD4 and RING finger domain 1 (UHRF1) was originally isolated in a yeast one-hybrid screen as a protein that binds to the CCAAT box within the topoisomerase II ␣ gene promoter [1]
In an effort to screen for small molecules that affect UHRF1 stability, we identified the HSP90 inhibitor 17-AAG as a potent inducer of UHRF1 degradation
Identification of HSP90 Inhibitor 17-AAG as a Small Molecule Potently Inducing Down-regulation of UHRF1 Proteins— Because UHRF1 is aberrantly highly expressed in multiple types of cancers and UHRF1 knockdown causes cell cycle arrest, activation of DNA damage response, and apoptosis in different types of cancer cells [16, 17], UHRF1 has been considered as a druggable target for cancer therapy [24, 25]

Summary

Results

Identification of HSP90 Inhibitor 17-AAG as a Small Molecule Potently Inducing Down-regulation of UHRF1 Proteins— Because UHRF1 is aberrantly highly expressed in multiple types of cancers and UHRF1 knockdown causes cell cycle arrest, activation of DNA damage response, and apoptosis in different types of cancer cells [16, 17], UHRF1 has been considered as a druggable target for cancer therapy [24, 25]. We generated HeLa cell lines stably expressing FLAG-tagged wild-type UHRF1 (WT), UHRF1 with deletion of RING domain (⌬RING), or UHRF1 with deletion of RING plus the polybasic region between SRA and RING (residues 1– 675) (Fig. 4D) These cells were treated with or without 17-DMAG in the presence or absence of the proteasome inhibitor MG132 for 24 h, and the levels of FLAGtagged UHRF1 and deletion mutants were determined by Western blotting analysis. To test this possibility, we made use of two different shRNAs to stably knock down CUL1, the common subunit of the SCF␤-TRCP E3 ligase in HEK293T and HeLa cell lines (Fig. 5E).

Discussion

Experimental Procedures

ADDITIONS AND CORRECTIONS