SPOP-mediated ubiquitination and degradation of PDK1 suppresses AKT kinase activity and oncogenic functions.
SPOP-mediated ubiquitination and degradation of PDK1 suppresses AKT kinase activity and oncogenic functions.
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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
DOI：
10.1186/s12943-021-01397-5
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发表时间：
2021-08-05
期刊：
影响因子：
37.3
通讯作者：
Guo J
中科院分区：
文献类型：
--
作者：
Jiang Q;Zheng N;Bu L;Zhang X;Zhang X;Wu Y;Su Y;Wang L;Zhang X;Ren S;Dai X;Wu D;Xie W;Wei W;Zhu Y;Guo J
3-磷酸肌醇依赖性蛋白激酶-1（PDK 1）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。虽然对PDK 1下游底物如蛋白激酶B（AKT）和核糖体蛋白S6激酶β（S6 K）的调控已经很好地建立，但PDK 1的上游调控因子，特别是其降解物，尚未被确定。采用基于成簇规则间隔短回文重复序列（CRISPR）的E3连接酶筛选方法来鉴定用于降解PDK 1的E3泛素连接酶。采用免疫印迹、免疫沉淀和免疫荧光染色等方法检测PDK 1与SPOP蛋白的相互作用和定位。采用免疫组织化学（MHC）染色研究前列腺癌组织中PDK 1和SPOP的表达。进行体内和体外泛素化测定以测量SPOP对PDK 1的泛素化缀合。采用体外激酶试验和质谱法鉴定酪蛋白激酶1（CK 1）和糖原合成酶激酶3（GSK 3）介导的PDK 1磷酸化。PDK 1突变的生物学效应以及与SPOP突变的相关性通过集落形成、软琼脂试验和体内异种移植小鼠模型进行。我们发现PDK 1经历了SPOP介导的泛素化和随后的蛋白酶体依赖性降解。具体而言，SPOP以CK 1/GSK 3 β介导的磷酸化依赖性方式通过共有降解决定子直接结合PDK 1。在病理学上，前列腺癌患者相关的SPOP突变损害PDK 1降解，从而激活AKT激酶，导致肿瘤恶性。同时，PDK 1降解决定子周围或内部发生的突变，通过阻断SPOP与降解决定子的结合或抑制CK 1或GSK 3 β介导的PDK 1磷酸化，可显著逃避SPOP介导的PDK 1降解，并通过激活AKT激酶发挥潜在的致癌作用。我们的研究结果不仅揭示了E3连接酶SPOP对PDK 1的生理调节，而且还强调了SPOP功能丧失突变或PDK 1功能获得突变通过激活AKT激酶在肿瘤发生中的致癌作用。在线版本包含补充材料，可通过10.1186/s12943-021-01397-5获得。
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. The online version contains supplementary material available at 10.1186/s12943-021-01397-5.
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影响因子：
82.9
作者：
Dai X;Gan W;Li X;Wang S;Zhang W;Huang L;Liu S;Zhong Q;Guo J;Zhang J;Chen T;Shimizu K;Beca F;Blattner M;Vasudevan D;Buckley DL;Qi J;Buser L;Liu P;Inuzuka H;Beck AH;Wang L;Wild PJ;Garraway LA;Rubin MA;Barbieri CE;Wong KK;Muthuswamy SK;Huang J;Chen Y;Bradner JE;Wei W
通讯作者：
Wei W
影响因子：
16
作者：
Gan W;Dai X;Lunardi A;Li Z;Inuzuka H;Liu P;Varmeh S;Zhang J;Cheng L;Sun Y;Asara JM;Beck AH;Huang J;Pandolfi PP;Wei W
通讯作者：
Wei W
影响因子：
8
作者：
Li, C.;Ao, J.;Fu, J.;Lee, D-F;Xu, J.;Lonard, D.;O'Malley, B. W.
通讯作者：
O'Malley, B. W.
影响因子：
12.3
作者：
Heitzer E;Ulz P;Belic J;Gutschi S;Quehenberger F;Fischereder K;Benezeder T;Auer M;Pischler C;Mannweiler S;Pichler M;Eisner F;Haeusler M;Riethdorf S;Pantel K;Samonigg H;Hoefler G;Augustin H;Geigl JB;Speicher MR
通讯作者：
Speicher MR
影响因子：
50.3
作者：
Inuzuka H;Tseng A;Gao D;Zhai B;Zhang Q;Shaik S;Wan L;Ang XL;Mock C;Yin H;Stommel JM;Gygi S;Lahav G;Asara J;Xiao ZX;Kaelin WG Jr;Harper JW;Wei W
通讯作者：
Wei W