title: 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.
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.
authors.affiliation: From the Department of Molecular Pharmacology and Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute, City of Hope, Duarte, California 91010.
authors.forename: Ching-Ying
authors.initials: CY
authors.lastname: Kuo
authors.affiliation: From the Department of Molecular Pharmacology and.
authors.forename: Xu
authors.initials: X
authors.lastname: Li
authors.affiliation: the Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China.
authors.forename: Xiang-Qian
authors.initials: XQ
authors.lastname: Kong
authors.affiliation: the Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Luo
authors.affiliation: the Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, and.
authors.forename: Che-Chang
authors.initials: CC
authors.lastname: Chang
authors.affiliation: From the Department of Molecular Pharmacology and.
authors.forename: Yiyin
authors.initials: Y
authors.lastname: Chung
authors.affiliation: the Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, and.
authors.forename: Hsiu-Ming
authors.initials: HM
authors.lastname: Shih
authors.affiliation: the State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, 197 Ruijin II Road, Shanghai 200025, China kli@epigenetic.us.
authors.forename: Keqin Kathy
authors.initials: KK
authors.lastname: Li
authors.affiliation: From the Department of Molecular Pharmacology and Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute, City of Hope, Duarte, California 91010, dann@coh.org.
authors.forename: David K
authors.initials: DK
authors.lastname: Ann
mesh_terms: D020816:Amino Acid Motifs; D004249:DNA Damage; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D012097:Repressor Proteins / Q000235:genetics; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000076183:Tripartite Motif-Containing Protein 28
chemical_list: D009687:Nuclear Proteins; C114070:RNF4 protein, human; D012097:Repressor Proteins; D025842:SUMO-1 Protein; D014157:Transcription Factors; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28
keywords: ATM; Arginine-rich Motif; DNA Damage Response; KAP1; Phosphorylation; Protein-Protein Interaction; RNF4; STUbL; SUMOylation; Ubiquitylation (Ubiquitination)