Abstract SY07-02: Involvement of ubiquitin ligase RNF4 in the DNA damage response
Authors: Hay Ronald T. 
Published: August 2012

Abstract
The ubiquitin-like family of protein modifiers (which are collectively referred to as UBLs) have a similar folded structure and are covalently linked to Lys side chains in target proteins. These linked UBL molecules represent additional surfaces for protein-protein interactions, and such interactions can recruit a range of effectors that alter the fate of modified proteins. UBLs (for example, ubiquitin) can be attached to proteins as single entities (monomeric modification), as single entities at multiple sites (multiple monomeric modification) or as a chain (polymeric modification). These different modes of modification represent unique signals that can result in different biological outcomes. In the case of the small ubiquitin-like modifier (SUMO), both monomeric and polymeric addition to substrates has been documented, but distinct biological functions have only recently been ascribed to these modifications. Intensive investigation has revealed a role for SUMO in the control of gene expression, the maintenance of genome integrity, intra cellular transport, protein stability and many other biological processes. Sumoylation involves the formation of an isopeptide bond between the carboxy-terminal Gly of SUMO and the amino group of a Lys side chain in the target protein. This reaction occurs in an analogous fashion to ubiquitylation and requires an E1 SUMO-activating enzyme (SAE1 and SAE2), an E2 SUMO-conjugating enzyme (UBC9, also known as UBE2I) and usually an E3 SUMO ligase. In most cases, sumoylation occurs on a Lys residue in the consensus modification site ψ-Lys-X-Glu, where ψ is a hydrophobic residue and X is any amino acid. This process is highly regulated in vivo by the SUMO-specific proteases that process SUMO precursors and deconjugate SUMO from the substrate. Whereas lower eukaryotes have one form of SUMO (Smt3), mammals have three SUMO paralogues (SUMO1, SUMO2 and SUMO3). The processed forms of SUMO2 and SUMO3 are almost identical except for three amino acids in their amino-terminal regions, and it has not been possible to functionally differentiate them. Whereas SUMO1 does not form SUMO chains in vivo, SUMO2 and SUMO3 can form such chains because they possess internal consensus SUMO modification sites. However, SUMO1 can be linked to the end of SUMO2 and SUMO3 chains, which terminates chain growth and results in the formation of mixed SUMO chains. Sumoylation results in diverse responses because it recruits a range of effector proteins that have SUMO-interaction motifs (SIMs). SIMs usually contain three hydrophobic residues in a sequence of four amino acids ((V/I/L)-X-(V/I/L)-(V/I/L)). This hydrophobic core forms a β-strand that can insert in either orientation into a hydrophobic groove between an α-helix and a β-strand in SUMO. In many proteins a stretch of negatively charged amino acids that surround the hydrophobic core of the SIM contributes to their SUMO-binding affinity. Over 200 RING domain-containing proteins have been identified by sequence analysis of the human genome. They have the potential to function as E3 ubiquitin ligases by recruiting both the substrate and a ubiquitin-bound E2 enzyme and catalyzing formation of the isopeptide bond between ubiquitin and substrate. RING finger protein 4 (RNF4; also known as SNURF) was originally identified as a novel gene that encodes a RING finger protein, functions as a co-activator of the androgen receptor and colocalizes with SUMO1 in PML nuclear bodies. RNF4 contains four potential SIMs that are located in the N-terminal region. The presence of multiple SIMs allows RNF4 to interact with multiple SUMO molecules. Thus RNF4 binds weakly to monomeric and dimeric SUMO2 but binds strongly to SUMO2 chains. The binding preference of RNF4 for SUMO chains is reflected in its ability to efficiently ubiquitylate SUMO chains in a RING- and SIM-dependent manner. PML was originally identified in acute promyelocytic leukaemia (APL), in which it is fused to retinoic acid receptor-α (RARα). Fusion of PML to RARα causes leukaemia because it blocks differentiation of haematopoietic progenitor cells. In normal cells, PML is localized to a small number of large nuclear bodies, but in cells containing PML-RARα, the fusion protein is dispersed into a large number of small nuclear bodies. In the absence of RNF4, PML protein levels increase and the protein accumulates with SUMO1 and SUMO2/3 in PML nuclear bodies, suggesting that RNF4 is involved in the stability of these nuclear structures. APL can be treated by administering arsenic, that triggers rapid degradation of PML and PML-RARα. This arsenic induced proteasomal degradation of PML and PML-RARα is preceded by sumoylation of PML and the recruitment of SUMO1 and SUMO2/3 into PML bodies. In cells depleted of RNF4 arsenic treatment fails to induce the degradation of PML. Conjugation of SUMO chains on Lys160 in PML is essential for arsenic-induced PML degradation, as the mutation of this residue or the depletion of SUMO2/3 abrogate the response to arsenic. In vitro, RNF4 only ubiquitylates PML that has been previously modified with SUMO2/3. This supports the idea that SUMO chains can trigger ubiquitylation of SUMO-modified proteins in vivo. Here we demonstrate that RNF4 also has an important role to play in the response of mammalian cells to DNA damage. RNF4 depletion by siRNA in human cells, or by gene-targeting in DT40 cells, sensitized cells to DNA damage. Recruitment of RNF4 to DNA breaks required its RING and SIM domains, and expression of NBS1, MDC1, RNF8, 53BP1 and Brca1. However these factors did not require RNF4 for recruitment to damaged DNA although clearance of 53BP1 from damage foci was delayed. SILAC-based-proteomics revealed that Mdc1 was SUMO modified in response to ionizing radiation. As a consequence of SUMO modification Mdc1 recruited RNF4 to mediate ubiquitylation at DNA damage sites. Failure to recruit RNF4 resulted in defective loading of RPA and Rad51 onto single-stranded DNA indicating that the primary role of RNF4 is in homologous-recombination. Thus RNF4 is a novel DNA-damage-responsive protein that integrates SUMO modification and ubiquitin signaling and plays a critical role in the cellular response to genotoxic stress.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY07-02. doi:1538-7445.AM2012-SY07-02