Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States.
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, United States.
Biochemistry. 2021 Feb 9;60(5):357-364. doi: 10.1021/acs.biochem.0c00869. Epub 2021 Jan 20.
WWP1 is an E3 ubiquitin ligase that has been reported to target the tumor suppressor lipid phosphatase PTEN. K740N and N745S are recently identified germline variants of WWP1 that have been linked to PTEN-associated cancers [Lee, Y. R., et al. (2020) ]. These WWP1 variants have been suggested to release WWP1 from its native autoinhibited state, thereby promoting enhanced PTEN ubiquitination as a mechanism for driving cancer. Using purified proteins and enzymatic assays, we investigate the possibility that K740N and N745S WWP1 possess enhanced ubiquitin ligase activity and demonstrate that these variants are similar to the wild type (WT) in both autoubiquitination and PTEN ubiquitination. Furthermore, K740N and N745S WWP1 show dependencies similar to those of WT in terms of allosteric activation by an engineered ubiquitin variant, upstream E2 concentration, and substrate ubiquitin concentration. Transfected WWP1 WT and mutants demonstrate comparable effects on cellular PTEN levels. These findings challenge the idea that K740N and N745S WWP1 variants promote cancer by enhanced PTEN ubiquitination.
WWP1 是一种 E3 泛素连接酶，据报道其可靶向肿瘤抑制脂质磷酸酶 PTEN。K740N 和 N745S 是最近鉴定的 WWP1 种系变异体，与 PTEN 相关癌症有关 [Lee, Y. R., 等人。(2020)]。这些 WWP1 变体被认为可以将 WWP1 从其天然的自动抑制状态中释放出来，从而促进增强的 PTEN 泛素化，作为驱动癌症的机制。我们使用纯化的蛋白质和酶促测定法研究了 K740N 和 N745S WWP1 是否具有增强的泛素连接酶活性的可能性，并证明这些变体在自泛素化和 PTEN 泛素化方面与野生型 (WT) 相似。此外，K740N 和 N745S WWP1 在由工程化泛素变体、上游 E2 浓度和底物泛素浓度引起的变构激活方面与 WT 具有相似的依赖性。转染的 WWP1 WT 和突变体在细胞 PTEN 水平上表现出类似的影响。这些发现挑战了 K740N 和 N745S WWP1 变体通过增强的 PTEN 泛素化促进癌症的观点。
