Moser Bernhard, Hochreiter Bernhard, Basílio José, Gleitsmann Viola, Panhuber Anja, Pardo-Garcia Alan, Hoesel Bastian, Salzmann Manuel, Resch Ulrike, Noreen Mamoona, Schmid Johannes A
Institute of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Schwarzspanierstraße 17, 1090, Vienna, Austria.
Mol Cancer. 2021 Jan 18;20(1):16. doi: 10.1186/s12943-021-01308-8.
The IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKβ, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. While several connections between NF-κB signaling and the oncogene c-Myc have been shown, functional links between the signaling molecules are still poorly studied.
Molecular interactions were shown by co-immunoprecipitation and FRET microscopy. Phosphorylation of c-Myc was shown by kinases assays and its activity by improved reporter gene systems. CRISPR/Cas9-mediated gene knockout and chemical inhibition were used to block IKK activity. The turnover of c-Myc variants was determined by degradation in presence of cycloheximide and by optical pulse-chase experiments.. Immunofluorescence of mouse prostate tissue and bioinformatics of human datasets were applied to correlate IKKα- and c-Myc levels. Cell proliferation was assessed by EdU incorporation and apoptosis by flow cytometry.
We show that IKKα and IKKβ bind to c-Myc and phosphorylate it at serines 67/71 within a sequence that is highly conserved. Knockout of IKKα decreased c-Myc-activity and increased its T58-phosphorylation, the target site for GSK3β, triggering polyubiquitination and degradation. c-Myc-mutants mimicking IKK-mediated S67/S71-phosphorylation exhibited slower turnover, higher cell proliferation and lower apoptosis, while the opposite was observed for non-phosphorylatable A67/A71-mutants. A significant positive correlation of c-Myc and IKKα levels was noticed in the prostate epithelium of mice and in a variety of human cancers.
Our data imply that IKKα phosphorylates c-Myc on serines-67/71, thereby stabilizing it, leading to increased transcriptional activity, higher proliferation and decreased apoptosis.
IκB激酶（IKK）复合物由两种酶IKKα和IKKβ组成，是炎症转录因子NF-κB的主要激活剂，NF-κB在许多癌症中持续激活。虽然已经显示出NF-κB信号传导与癌基因c-Myc之间的几种联系，但信号分子之间的功能联系仍研究不足。
通过免疫共沉淀和荧光共振能量转移显微镜显示分子相互作用。通过激酶测定显示c-Myc的磷酸化，并通过改进的报告基因系统显示其活性。使用CRISPR/Cas9介导的基因敲除和化学抑制来阻断IKK活性。通过在放线菌酮存在下的降解和光脉冲追踪实验来确定c-Myc变体的周转。应用小鼠前列腺组织的免疫荧光和人类数据集的生物信息学来关联IKKα和c-Myc水平。通过EdU掺入评估细胞增殖，并通过流式细胞术评估细胞凋亡。
我们表明IKKα和IKKβ与c-Myc结合并在高度保守的序列中的丝氨酸67/71处使其磷酸化。IKKα的敲除降低了c-Myc活性并增加了其T58磷酸化，T58磷酸化是GSK3β的靶位点，触发多聚泛素化和降解。模拟IKK介导的S67/S71磷酸化的c-Myc突变体表现出较慢的周转、较高的细胞增殖和较低的细胞凋亡，而对于不可磷酸化的A67/A71突变体则观察到相反的情况。在小鼠的前列腺上皮和多种人类癌症中，注意到c-Myc和IKKα水平之间存在显著的正相关。
我们的数据表明IKKα在丝氨酸-67/71处使c-Myc磷酸化，从而使其稳定，导致转录活性增加、增殖增加和细胞凋亡减少。
