3-phosphoinositide-dependent protein kinase-1 (PDK1) acts as a master kinase of protein kinase A, G, and C family (AGC) kinase to predominantly govern cell survival, proliferation, and metabolic homeostasis. Although the regulations to PDK1 downstream substrates such as protein kinase B (AKT) and ribosomal protein S6 kinase beta (S6K) have been well established, the upstream regulators of PDK1, especially its degrader, has not been defined yet. A clustered regularly interspaced short palindromic repeats (CRISPR)-based E3 ligase screening approach was employed to identify the E3 ubiquitin ligase for degrading PDK1. Western blotting, immunoprecipitation assays and immunofluorescence (IF) staining were performed to detect the interaction or location of PDK1 with speckle-type POZ protein (SPOP). Immunohistochemistry (IHC) staining was used to study the expression of PDK1 and SPOP in prostate cancer tissues. In vivo and in vitro ubiquitination assays were performed to measure the ubiquitination conjugation of PDK1 by SPOP. In vitro kinase assays and mass spectrometry approach were carried out to identify casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)-mediated PDK1 phosphorylation. The biological effects of PDK1 mutations and correlation with SPOP mutations were performed with colony formation, soft agar assays and in vivo xenograft mouse models. We identified that PDK1 underwent SPOP-mediated ubiquitination and subsequent proteasome-dependent degradation. Specifically, SPOP directly bound PDK1 by the consensus degron in a CK1/GSK3β-mediated phosphorylation dependent manner. Pathologically, prostate cancer patients associated mutations of SPOP impaired PDK1 degradation and thus activated the AKT kinase, resulting in tumor malignancies. Meanwhile, mutations that occurred around or within the PDK1 degron, by either blocking SPOP to bind the degron or inhibiting CK1 or GSK3β-mediated PDK1 phosphorylation, could markedly evade SPOP-mediated PDK1 degradation, and played potently oncogenic roles via activating the AKT kinase. Our results not only reveal a physiological regulation of PDK1 by E3 ligase SPOP, but also highlight the oncogenic roles of loss-of-function mutations of SPOP or gain-of-function mutations of PDK1 in tumorigenesis through activating the AKT kinase.

中文翻译：

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SPOP 介导的 PDK1 泛素化和降解抑制 AKT 激酶活性和致癌功能

3-磷酸​​肌醇依赖性蛋白激酶-1 (PDK1) 作为蛋白激酶 A、G 和 C 家族 (AGC) 激酶的主激酶，主要控制细胞存活、增殖和代谢稳态。尽管对 PDK1 下游底物如蛋白激酶 B (AKT) 和核糖体蛋白 S6 激酶 β (S6K) 的监管已经确立，但 PDK1 的上游调节剂，尤其是其降解剂，尚未确定。采用基于聚集规则间隔短回文重复 (CRISPR) 的 E3 连接酶筛选方法来鉴定用于降解 PDK1 的 E3 泛素连接酶。进行蛋白质印迹、免疫沉淀测定和免疫荧光 (IF) 染色以检测 PDK1 与斑点型 POZ 蛋白 (SPOP) 的相互作用或位置。免疫组织化学 (IHC) 染色用于研究 PDK1 和 SPOP 在前列腺癌组织中的表达。进行体内和体外泛素化测定以测量 SPOP 对 PDK1 的泛素化结合。进行体外激酶测定和质谱方法以鉴定酪蛋白激酶 1 (CK1) 和糖原合酶激酶 3 (GSK3) 介导的 PDK1 磷酸化。PDK1 突变的生物学效应和与 SPOP 突变的相关性通过集落形成、软琼脂测定和体内异种移植小鼠模型进行。我们发现 PDK1 经历了 SPOP 介导的泛素化和随后的蛋白酶体依赖性降解。具体来说，SPOP 通过共有 degron 以 CK1/GSK3β 介导的磷酸化依赖性方式直接结合 PDK1。病理上，前列腺癌患者相关的 SPOP 突变损害了 PDK1 的降解，从而激活了 AKT 激酶，导致肿瘤恶性肿瘤。同时，PDK1 degron 周围或内部发生的突变，通过阻断 SPOP 与 degron 结合或抑制 CK1 或 GS​​K3β 介导的 PDK1 磷酸化，可以显着逃避 SPOP 介导的 PDK1 降解，并通过激活 AKT 激酶发挥有效的致癌作用。我们的研究结果不仅揭示了 E3 连接酶 SPOP 对 PDK1 的生理调节，而且突出了 SPOP 功能丧失突变或 PDK1 功能获得突变通过激活 AKT 激酶在肿瘤发生中的致癌作用。PDK1 degron 周围或内部发生的突变，通过阻断 SPOP 与 degron 结合或抑制 CK1 或 GS​​K3β 介导的 PDK1 磷酸化，可以显着逃避 SPOP 介导的 PDK1 降解，并通过激活 AKT 激酶发挥有效的致癌作用。我们的研究结果不仅揭示了 E3 连接酶 SPOP 对 PDK1 的生理调节，而且突出了 SPOP 功能丧失突变或 PDK1 功能获得突变通过激活 AKT 激酶在肿瘤发生中的致癌作用。PDK1 degron 周围或内部发生的突变，通过阻断 SPOP 与 degron 结合或抑制 CK1 或 GS​​K3β 介导的 PDK1 磷酸化，可以显着逃避 SPOP 介导的 PDK1 降解，并通过激活 AKT 激酶发挥有效的致癌作用。我们的研究结果不仅揭示了 E3 连接酶 SPOP 对 PDK1 的生理调节，而且突出了 SPOP 功能丧失突变或 PDK1 功能获得突变通过激活 AKT 激酶在肿瘤发生中的致癌作用。