Macroautophagy/autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNA-dependent protein kinase PRKDC/DNA-PKcs as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed PRKDC upstream of the AMP-dependent protein kinase (AMPK) complex and ULK1 kinase. In normal culture conditions, PRKDC interacted with the AMPK complex and phosphorylated its nucleotide-sensing γ1 subunit PRKAG1/AMPKγ1 at Ser192 and Thr284, both events being significantly reduced upon the activation of the AMPK complex. Alanine substitutions of PRKDC phosphorylation sites in PRKAG1 reduced AMPK complex activation without affecting its nucleotide sensing capacity. Instead, the disturbance of PRKDC-mediated phosphorylation of PRKAG1 inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with STK11 (serine/threonine kinase 11). Taken together, our data suggest that PRKDC-mediated phosphorylation of PRKAG1 primes AMPK complex to the lysosomal activation by STK11 in cancer cells thereby linking DNA damage response to autophagy and cellular metabolism.

Abbreviations: AXIN1: axin 1; 3-MA: 3-methyladenine; 5-FU: 5-fluorouracil; AA mutant: double alanine mutant (S192A, T284A) of PRKAG1; ACACA: acetyl-CoA carboxylase alpha; AICAR: 5-Aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ATM: ataxia telangiectasia mutated; ATR: ATM serine/threonine kinase; AV: autophagic vacuole; AVd: degradative autophagic vacuole; AVi: initial autophagic vacuole; BECN1: beclin 1; BSA: bovine serum albumin; CBS: cystathionine beta-synthase; CDK7: cyclin dependent kinase 7; CDKN1A: cyclin dependent kinase inhibitor 1A; EGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S transferase; H2AX/H2AFX: H2A.X variant histone; HBSS: Hanks balanced salt solution; IP: immunopurification; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K9: mitogen-activated protein kinase kinase kinase 9; mRFP: monomeric red fluorescent protein; mCh: mCherry; MCM7: minichromosome maintenance complex component 7; MTORC1: mechanistic target of rapamycin kinase complex 1; NHEJ: non-homologous end joining; NRBP2: nuclear receptor binding protein 2; NTC: non-targeting control; NUAK1: NUAK family kinase 1; PBS: phosphate-buffered saline; PIK3AP1: phosphoinositide-3-kinase adaptor protein 1; PIK3CA: phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha; PIKK: phosphatidylinositol 3-kinase-related kinase; PRKAA: protein kinase AMP-activated catalytic subunit alpha; PRKAB: protein kinase AMP-activated non-catalytic subunit beta; PRKAG: protein kinase AMP-activated non-catalytic subunit gamma; PRKDC: protein kinase, DNA-activated, catalytic subunit; RLuc: Renilla luciferase; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TP53: tumor protein p53; TSKS: testis specific serine kinase substrate; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.

巨自噬/自噬是细胞保护性细胞应激反应的核心组成部分。为了阐明应激诱导的自噬信号传导，我们筛选了人类激酶组 siRNA 文库，用于 MCF7 人乳腺癌细胞中自噬通量的调节因子，并鉴定了 DNA 依赖性蛋白激酶 PRKDC/DNA-PKcs 的催化亚基作为基础和 DNA 的正调节因子损伤诱导的自噬。自噬调节信号级联分析将 PRKDC 置于 AMP 依赖性蛋白激酶 (AMPK) 复合物和 ULK1 激酶的上游。在正常培养条件下，PRKDC 与 AMPK 复合物相互作用，并在 Ser192 和 Thr284 位点磷酸化其核苷酸感应 γ1 亚基 PRKAG1/AMPKγ1，这两种事件在 AMPK 复合物激活后显着减少。PRKAG1 中 PRKDC 磷酸化位点的丙氨酸取代减少了 AMPK 复合物的激活，而不影响其核苷酸传感能力。相反，PRKDC 介导的 PRKAG1 磷酸化的干扰抑制了 AMPK 复合物的溶酶体定位及其与 STK11（丝氨酸/苏氨酸激酶 11）的饥饿诱导关联。总之，我们的数据表明，PRKDC 介导的 PRKAG1 磷酸化使 AMPK 复合物引发癌细胞中 STK11 的溶酶体活化，从而将 DNA 损伤反应与自噬和细胞代谢联系起来。

缩写：轴1：轴1；3-MA：3-甲基腺嘌呤；5-FU：5-氟尿嘧啶；AA突变体：PRKAG1的双丙氨酸突变体（S192A、T284A）；ACACA：乙酰辅酶A羧化酶α；AICAR：5-氨基咪唑-4-甲酰胺核糖核苷酸；AMPK：AMP活化蛋白激酶；ATG：自噬相关；ATM：共济失调毛细血管扩张症突变；ATR：ATM丝氨酸/苏氨酸激酶；AV：自噬泡；AVd：降解自噬泡；AVi：初始自噬泡；BECN1: beclin 1; BSA：牛血清白蛋白；CBS：胱硫醚β-合酶；CDK7：细胞周期蛋白依赖性激酶 7；CDKN1A：细胞周期蛋白依赖性激酶抑制剂 1A；EGFP：增强型绿色荧光蛋白；GAPDH：3-磷酸甘油醛脱氢酶；GST：谷胱甘肽S转移酶；H2AX/H2AFX：H2A.X 变异组蛋白；HBSS：汉克斯平衡盐溶液；IP：免疫纯化；IR：电离辐射；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；MAP3K9：丝裂原活化蛋白激酶激酶激酶 9；mRFP：单体红色荧光蛋白；mCh：mCherry；MCM7：微染色体维持复合物组分7；MTORC1：雷帕霉素激酶复合物 1 的机制靶点；NHEJ：非同源末端连接；NRBP2：核受体结合蛋白2；NTC：非靶向控制；NUAK1：NUAK家族激酶1；PBS：磷酸盐缓冲盐水；PIK3AP1：磷酸肌醇-3-激酶衔接蛋白 1；PIK3CA：磷脂酰肌醇-4,5-二磷酸3-激酶催化亚基α；PIKK：磷脂酰肌醇 3-激酶相关激酶；PRKAA：蛋白激酶 AMP 激活的催化亚基 α；PRKAB：蛋白激酶 AMP 激活的非催化亚基β；PRKAG：蛋白激酶 AMP 激活的非催化亚基 γ；PRKDC：蛋白激酶，DNA 激活，催化亚基；RLuc：海肾荧光素酶；RPS6KB1：核糖体蛋白S6激酶B1；SQSTM1：螯合体 1；STK11/LKB1：丝氨酸/苏氨酸激酶 11；TP53：肿瘤蛋白p53；TSKS：睾丸特异性丝氨酸激酶底物；ULK1：unc-51 类似自噬激活激酶 1；WIPI2：WD 重复结构域，磷酸肌醇相互作用 2；WT：野生型。