An Arginine-rich Motif of Ring Finger Protein 4 (RNF4) Oversees the Recruitment and Degradation of the Phosphorylated and SUMOylated Krüppel-associated Box Domain-associated Protein 1 (KAP1)/TRIM28 Protein during Genotoxic Stress

Ching-Ying Kuo,Xu Li,Xiang-Qian Kong,Cheng Luo,Che-Chang Chang,Yiyin Chung,Hsiu-Ming Shih,Keqin Kathy Li,David K Ann

doi:10.1074/jbc.m114.555672

Abstract

Krüppel-associated box domain-associated protein 1 (KAP1) is a universal transcriptional corepressor that undergoes multiple posttranslational modifications (PTMs), including SUMOylation and Ser-824 phosphorylation. However, the functional interplay of KAP1 PTMs in regulating KAP1 turnover during DNA damage response remains unclear. To decipher the role and cross-talk of multiple KAP1 PTMs, we show here that DNA double strand break-induced KAP1 Ser-824 phosphorylation promoted the recruitment of small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, ring finger protein 4 (RNF4), and subsequent RNF4-mediated, SUMO-dependent degradation. Besides the SUMO interacting motif (SIM), a previously unrecognized, but evolutionarily conserved, arginine-rich motif (ARM) in RNF4 acts as a novel recognition motif for selective target recruitment. Results from combined mutagenesis and computational modeling studies suggest that RNF4 utilizes concerted bimodular recognition, namely SIM for Lys-676 SUMOylation and ARM for Ser(P)-824 of simultaneously phosphorylated and SUMOylated KAP1 (Ser(P)-824-SUMO-KAP1). Furthermore, we proved that arginines 73 and 74 within the ARM of RNF4 are required for efficient recruitment to KAP1 or accelerated degradation of promyelocytic leukemia protein (PML) under stress. In parallel, results of bimolecular fluorescence complementation assays validated the role of the ARM in recognizing Ser(P)-824 in living cells. Taken together, we establish that the ARM is required for RNF4 to efficiently target Ser(P)-824-SUMO-KAP1, conferring ubiquitin Lys-48-mediated proteasomal degradation in the context of double strand breaks. The conservation of such a motif may possibly explain the requirement for timely substrate selectivity determination among a myriad of SUMOylated proteins under stress conditions. Thus, the ARM dynamically regulates the SIM-dependent recruitment of targets to RNF4, which could be critical to dynamically fine-tune the abundance of Ser(P)-824-SUMO-KAP1 and, potentially, other SUMOylated proteins during DNA damage response.

Highlights

The function of Krüppel-associated box domain-associated protein 1 (KAP1) is regulated by multiple posttranslational modifications during DNA damage response
We aimed to explore the contribution of each KAP1 posttranslational modifications (PTMs), namely phosphorylation, SUMOylation, and ubiquitylation, to the temporal regulation of KAP1 turnover
We found that IR induced a transient appearance of Ser(P)-824-KAP1 in the absence of CHX (Fig. 1A, top left panel) and that there was a correlation between decreased ring finger protein 4 (RNF4) and increased Ser(P)824-KAP1 signals at later time points in the CHX-treated, IRexposed MCF7 cells compared with those in the vehicletreated, IR-exposed counterparts (Fig. 1A, top right panel)

Summary

Introduction

The function of KAP1 is regulated by multiple posttranslational modifications during DNA damage response. To decipher the role and cross-talk of multiple KAP1 PTMs, we show here that DNA double strand break-induced KAP1 Ser-824 phosphorylation promoted the recruitment of small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, ring finger protein 4 (RNF4), and subsequent RNF4-mediated, SUMO-dependent degradation. We establish that the ARM is required for RNF4 to efficiently target Ser(P)-824-SUMO-KAP1, conferring ubiquitin Lys-48-mediated proteasomal degradation in the context of double strand breaks. The conservation of such a motif may possibly explain the requirement for timely substrate selectivity determination among a myriad of SUMOylated proteins under stress conditions. The ARM dynamically regulates the SIM-dependent recruitment of targets to RNF4, which could be critical to dynamically fine-tune the abundance of Ser(P)-824-SUMOKAP1 and, potentially, other SUMOylated proteins during DNA damage response

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Results

Conclusion