PO.MCB01.01 · 分子与细胞生物学
CDK2调控前列腺癌细胞中RIPK2的泛素化和稳定性
CDK2 controls RIPK2 ubiquitination and stability in prostate cancer cells
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：前列腺癌(PC)转移驱动PC相关的死亡率和发病率，然而这一致命过程背后的分子机制仍未完全阐明。此前，我们证明RIPK2对PC进展和转移至关重要(Nat. Commun., 2022)。然而，控制PC细胞中RIPK2蛋白水平的调控机制仍不清楚。本研究旨在通过研究PC细胞中RIPK2泛素化和降解的调控来填补这一知识空白，特别关注CDK2，我们将其鉴定为RIPK2调控c-Myc癌蛋白的关键介质。
方法：用两种经临床评估的CDK2选择性抑制剂INX-315和PF-07104091处理PC细胞——22Rv1(雄激素受体阳性)和PC3(雄激素受体阴性)。通过Western blotting测量RIPK2蛋白水平的变化。使用放线菌酮(CHX)追踪试验评估RIPK2稳定性，并用MG132处理评估蛋白酶体降解。通过免疫沉淀，然后用K48连接泛素抗体进行Western blotting，检测RIPK2的K48连接泛素化。通过血清饥饿或用Ro-3306、羟基脲、胸苷或monastrol处理实现细胞周期同步化。使用邻近连接试验(PLA)评估RIPK2与CDK2、其E3连接酶ZNRF4，或去泛素化酶YOD1、OTUB2和OTUB5之间的蛋白-蛋白相互作用。使用非配对双尾学生t检验进行统计分析。
结果：CDK2抑制以蛋白酶体依赖的方式显著降低了RIPK2蛋白半衰期，并增加了RIPK2的K48连接泛素化。RIPK2蛋白丰度在G2期达到峰值，滞后于活性CDK2(其稳定化激酶)的S期峰值。邻近连接试验显示，RIPK2主要在细胞质中与CDK2、ZNRF4、YOD1和OTUB5结合。值得注意的是，CDK2抑制以剂量依赖的方式破坏了RIPK2与YOD1的结合，但不破坏与ZNRF4的结合，提示YOD1参与CDK2介导的RIPK2稳定性控制。总之，这些发现将CDK2鉴定为PC细胞中RIPK2泛素化和稳定性的关键调节因子。
结论：激酶活性的CDK2抑制RIPK2的K48连接泛素化，并保护其免受蛋白酶体降解。这种保护可能通过调控PC细胞中RIPK2与其去泛素化酶YOD1之间的结合来介导。这些发现支持进一步研究CDK2在PC和其他癌症类型中稳定RIPK2的分子机制。*通讯作者：Wei.Yang@stonybrook.edu
查看英文原文 English abstract
Background: Prostate cancer (PC) metastasis drives PC-related mortality and morbidity, yet the molecular mechanisms underlying this lethal process remains incompletely understood. Previously, we demonstrated that RIPK2 is critical for PC progression and metastasis ( Nat. Commun. , 2022). However, the regulatory mechanisms controlling RIPK2 protein levels in PC cells remain unclear. This study aims to address this knowledge gap by investigating the regulation of RIPK2 ubiquitination and degradation in PC cells, with a particular focus on CDK2, which we identified as a key mediator of RIPK2 regulation of the c-Myc oncoprotein.
Methods : PC cells-22Rv1 (androgen receptor-positive) and PC3 (androgen receptor-negative)-were treated with two clinically evaluated, CDK2-selective inhibitors, INX-315 and PF-07104091. Changes in RIPK2 protein levels were measured by western blotting. RIPK2 stability was assessed using cycloheximide (CHX) chase assays, and proteasomal degradation was evaluated with MG132 treatment. K48-linked ubiquitination of RIPK2 was examined by immunoprecipitation followed by western blotting with K48-linked ubiquitin antibodies. Cell cycle synchronization was achieved by serum starvation or treatment with Ro-3306, hydroxyurea, thymidine, or monastrol. Proximity ligation assays (PLA) were used to assess protein-protein interactions between RIPK2 and CDK2, its E3 ligase ZNRF4, or deubiquitinases YOD1, OTUB2, and OTUB5. Statistical analyses were performed using unpaired, two-tailed Student's t -tests.
Results: CDK2 inhibition significantly reduced RIPK2 protein half-life in a proteasome-dependent manner and increased RIPK2 K48-linked ubiquitination. RIPK2 protein abundance peaks in the G2 phase, lagging behind the S-phase peak of active CDK2, its stabilizing kinase. Proximity ligation assays showed that RIPK2 associates with CDK2, ZNRF4, YOD1, and OTUB5 primarily in the cytoplasm. Notably, CDK2 inhibition disrupted RIPK2 association with YOD1 but not ZNRF4 in a dose-dependent manner, implicating YOD1 in CDK2-mediated control of RIPK2 stability. Together, these findings identify CDK2 as a key regulator of RIPK2 ubiquitination and stability in PC cells.
Conclusion : Kinase-active CDK2 inhibits the K48-linked ubiquitination of RIPK2 and protects it from proteasomal degradation. This protection is potentially mediated by regulating the association between RIPK2 and its deubiquitinase YOD1 in PC cells. The findings support further investigation into the molecular mechanisms by which CDK2 stabilizes RIPK2 in PC and other cancer types. *Corresponding Author: Wei.Yang@stonybrook.edu
利益披露 Disclosure
L. M. Guerra, None..
A. M. Elgehama, None..
J. G. P. Sanches, None..
L. Chiu, None..
W. Yang*, None.