Regulation of CYLD activity and specificity by phosphorylation and ubiquitin-binding CAP-Gly domains

Non-degradative ubiquitin chains and phosphorylation events govern signaling responses by innate immune receptors. The deubiquitinase CYLD in complex with SPATA2 is recruited to receptor signaling complexes by the ubiquitin ligase LUBAC and regulates Met1- and Lys63-linked polyubiquitin and receptor signaling outcomes. Here, we investigated the molecular determinants of CYLD activity. We reveal that two CAP-Gly domains in CYLD are ubiquitin binding domains and demonstrate a requirement of CAP-Gly3 for CYLD activity and regulation of immune receptor signaling. Moreover, we identify a phosphorylation switch outside of the catalytic USP domain, which activates CYLD selectively towards Lys63-linked polyubiquitin. The phosphorylated residue Ser568 is a novel TNF-regulated phosphorylation site in CYLD and works in concert with Ser418 to enable CYLD-mediated deubiquitination and immune receptor signaling. We propose that phosphorylated CYLD together with SPATA2 and LUBAC functions as a ubiquitin-editing complex that rebalances Lys63- and Met1-linked polyubiquitin at receptor signaling complexes, resulting in escalated LUBAC signaling.

非降解型泛素链与磷酸化事件共同调控天然免疫受体的信号应答。与SPATA2形成复合物的去泛素化酶CYLD可被泛素连接酶LUBAC招募至受体信号复合体，并调控甲硫氨酸1位（Met1）与赖氨酸63位（Lys63）连接的多聚泛素链，进而决定受体信号转导的结局。本研究针对CYLD活性的分子决定因素展开了系统探究。我们发现CYLD中存在两个CAP-甘氨酸（CAP-Gly）结构域，其可作为泛素结合结构域；同时证实CAP-Gly3结构域是CYLD发挥活性、调控免疫受体信号转导所必需的元件。此外，我们在泛素特异性蛋白酶（USP）催化结构域外发现了一处磷酸化开关，该开关可选择性激活CYLD对赖氨酸63位（Lys63）连接的多聚泛素链的水解活性。磷酸化位点Ser568是CYLD中一处受肿瘤坏死因子（TNF）调控的新型磷酸化位点，其可与Ser418协同作用，介导CYLD依赖的去泛素化过程与免疫受体信号转导。我们提出，磷酸化后的CYLD可与SPATA2、LUBAC共同构成泛素编辑复合体，该复合体可在受体信号复合体处重新平衡赖氨酸63位与甲硫氨酸1位连接的多聚泛素链水平，最终增强LUBAC介导的信号转导。