An Arginine-rich Motif of Ring Finger Protein 4 (RNF4) Oversees the Recruitment and Degradation of the Phosphorylated and SUMOylated Kruppel-associated Box Domain-associated Protein 1 (KAP1)/TRIM28 Protein during Genotoxic Stress
An Arginine-rich Motif of Ring Finger Protein 4 (RNF4) Oversees the Recruitment and Degradation of the Phosphorylated and SUMOylated Kruppel-associated Box Domain-associated Protein 1 (KAP1)/TRIM28 Protein during Genotoxic Stress
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DOI：
10.1074/jbc.m114.555672
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发表时间：
2014-07-25
影响因子：
4.8
通讯作者：
Ann, David K.
中科院分区：
文献类型：
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作者：
Kuo, Ching-Ying;Li, Xu;Ann, David K.
Kruppel相关的BOX结构域相关蛋白1(KAP1)是一种通用的转录辅阻遏子，它经历了多种翻译后修饰(PTM)，包括SUMO化和Ser-824磷酸化。然而，KAP1PTMS在DNA损伤反应过程中调节KAP1周转的功能相互作用尚不清楚。为了解释多个KAP1 PTM的作用和相互作用，我们在这里展示了DNA双链断裂诱导的KAP1Ser-824磷酸化促进小泛素样修饰物(SUMO)靶向泛素E3连接酶、RNF4(RNF4)的募集，以及随后RNF4介导的相扑依赖的降解。除了相扑相互作用基序(SIM)外，RNF4中一个先前未被识别但在进化上保守的富含精氨酸的基序(ARM)作为一种新的识别基序用于选择性目标招募。联合突变和计算模型研究的结果表明，RNF4利用了协同的双模式识别，即SIM用于Lys-676的SUMO化，ARM用于同时磷酸化和SUMO化的KAP1的Ser(P)-824(Ser(P)-824-SUMO-KAP1)。此外，我们还证明了RNF4臂中的精氨酸73和74是在应激状态下有效地募集到KAP1或加速早幼粒细胞白血病蛋白(PML)降解所必需的。同时，双分子荧光互补分析的结果证实了ARM在识别活细胞中的Ser(P)-824方面的作用。综上所述，我们确定RNF4需要手臂来有效靶向Ser(P)-824-SUMO-KAP1，从而在双链断裂的背景下实现泛素Lys-48介导的蛋白酶体降解。这种基序的保守性可能解释了在胁迫条件下对大量SUMO化蛋白质进行适时底物选择性测定的必要性。因此，ARM动态地调节SIM依赖的靶向RNF4的招募，这对于在DNA损伤反应中动态微调Ser(P)-824-SUMO-KAP1和潜在的其他SUMO化蛋白的丰度至关重要。
Kruppel-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.