ABSTRACT

MLKL (mixed lineage kinase domain like pseudokinase) is a well-known core component of necrosome that executes necroptotic cell death upon phosphorylation by RIPK3 (receptor interacting serine/threonine kinase 3). Recent studies also implicate a role of MLKL in endosomal trafficking, which is not always dependent on RIPK3. Using mouse Neuro-2a and L929 as well as human HEK293 and HT29 cells, we show here that MLKL is phosphorylated in response to serum and amino acid deprivation from the culture medium, in a manner that depends on CAMK2/CaMKII (calcium/calmodulin dependent protein kinase II) but not RIPK3. The starvation-induced increase in MLKL phosphorylation was accompanied by decreases in levels of lipidated MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; LC3-II) and SQSTM1/p62 (sequestosome 1), markers of autophagosomes. These changes were prevented by disrupting either MLKL or CAMK2 by pharmacology and genetic manipulations. Moreover, disrupting MLKL or CAMK2 also inhibited the incorporation of LC3-II into autolysosomes, demonstrating a role of the CAMK2-MLKL pathway in facilitating autophagic flux during short-term starvation, in contrast to necroptosis which suppressed autophagic flux. Furthermore, unlike the necroptotic pathway, the starvation-evoked CAMK2-mediated MLKL phosphorylation protected cells from starvation-induced death. We propose that upon nutrient deprivation, MLKL is activated by CAMK2, which in turn facilitates membrane scission needed for autophagosome maturation, allowing the proper fusion of the autophagosome with lysosome and the subsequent substance degradation. This novel function is independent of RIPK3 and is not involved in necroptosis, implicating new roles for this pseudokinase in cell survival, signaling and metabolism.

Abbreviations: CAMK2/CaMKII: calcium/calmodulin dependent protein kinase II; DIABLO/SMAC: direct inhibitor of apoptosis-binding protein with low pI/second mitochondria-derived activator of caspase; ECS: extracellular solution; ESCRT: endosomal sorting complexes required for transport; FBS: fetal bovine serum; GSK3B: glycogen synthase kinase 3 beta; HBSS: Hanks’ balanced salt solution; KO: knockout; LC3-II: lipidated microtubule associated protein 1 light chain 3 beta; LDH: lactate dehydrogenase; MLKL: mixed lineage kinase domain like pseudokinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; N2a: Neuro-2a neuroblastoma; Nec-1: necrostatin-1; NSA: necrosulfonamide; PBS: phosphate-buffered saline; PI: propidium iodide; PK-hLC3: pHluorin-mKate2-human LC3; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; ROS: reactive oxygen species; RPS6KB1/S6K: ribosomal protein S6 kinase B1; shRNA: short hairpin RNA; siRNA: small interference RNA; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TSZ, treatment with TNF + DIABLO mimetics + z-VAD-FMK.

摘要

MLKL（混合谱系激酶结构域，如假激酶）是一种众所周知的坏死体核心成分，它在被 RIPK3（受体相互作用的丝氨酸/苏氨酸激酶 3）磷酸化后执行坏死性细胞死亡。最近的研究还暗示 MLKL 在内涵体运输中的作用，这并不总是依赖于 RIPK3。使用小鼠 Neuro-2a 和 L929 以及人类 HEK293 和 HT29 细胞，我们在此显示 MLKL 以依赖于 CAMK2/CaMKII（钙/钙调蛋白依赖性）的方式磷酸化以响应培养基中血清和氨基酸的剥夺蛋白激酶 II) 但不是 RIPK3。饥饿诱导的 MLKL 磷酸化增加伴随着自噬体标志物脂化 MAP1LC3B/LC3B（微管相关蛋白 1 轻链 3 β；LC3-II）和 SQSTM1/p62（sequestosome 1）水平的降低。通过药理学和基因操作破坏 MLKL 或 CAMK2 可以防止这些变化。此外，破坏 MLKL 或 CAMK2 还抑制了 LC3-II 掺入自溶酶体中，证明了 CAMK2-MLKL 途径在促进短期饥饿期间自噬通量的作用，这与抑制自噬通量的坏死性凋亡形成对比。此外，与坏死性凋亡途径不同，饥饿诱发的 CAMK2 介导的 MLKL 磷酸化保护细胞免于饥饿诱导的死亡。我们提出，在营养剥夺后，MLKL 被 CAMK2 激活，这反过来又促进了自噬体成熟所需的膜断裂，从而使自噬体与溶酶体适当融合并随后进行物质降解。

缩写：CAMK2/CaMKII：钙/钙调蛋白依赖性蛋白激酶 II；DIABLO/SMAC：凋亡结合蛋白的直接抑制剂，具有低 pI/第二个线粒体衍生的半胱天冬酶激活剂；ECS：细胞外溶液；ESCRT：运输所需的内体分选复合物；FBS：胎牛血清；GSK3B：糖原合酶激酶 3 β；HBSS：汉克斯平衡盐溶液；KO：淘汰赛；LC3-II：脂化微管相关蛋白1轻链3β；LDH：乳酸脱氢酶；MLKL：混合谱系激酶结构域，如假激酶；MTOR：雷帕霉素激酶的机制靶点；MTORC1：MTOR复合物1；N2a：Neuro-2a 神经母细胞瘤；Nec-1：necrostatin-1；NSA：坏死性磺胺；PBS：磷酸盐缓冲液；PI：碘化丙啶；PK-hLC3：pHluorin-mKate2-人 LC3；RIPK1：受体相互作用的丝氨酸/苏氨酸激酶 1；RIPK3：受体相互作用的丝氨酸/苏氨酸激酶 3；ROS：活性氧；RPS6KB1/S6K：核糖体蛋白 S6 激酶 B1；shRNA：短发夹RNA；siRNA：小干扰RNA；SQSTM1/p62：隔离体 1；TBS：Tris 缓冲盐水；TNF/TNF-α：肿瘤坏死因子；TSZ，用 TNF + DIABLO 模拟物 + z-VAD-FMK 治疗。