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CAMK2/CaMKII activates MLKL in short-term starvation to facilitate autophagic flux. 2022

Qionghui Zhan, and Jaepyo Jeon, and Ying Li, and Yu Huang, and Jian Xiong, and Qiaochu Wang, and Tian-Le Xu, and Yong Li, and Fu-Hai Ji, and Guangwei Du, and Michael X Zhu
Department of Anesthesiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.

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.

UI MeSH Term Description Entries
D008869 Microtubule-Associated Proteins High molecular weight proteins found in the MICROTUBULES of the cytoskeletal system. Under certain conditions they are required for TUBULIN assembly into the microtubules and stabilize the assembled microtubules. Ensconsin,Epithelial MAP, 115 kDa,Epithelial Microtubule-Associate Protein, 115 kDa,MAP4,Microtubule Associated Protein,Microtubule Associated Protein 4,Microtubule Associated Protein 7,Microtubule-Associated Protein,Microtubule-Associated Protein 7,E-MAP-115,MAP1 Microtubule-Associated Protein,MAP2 Microtubule-Associated Protein,MAP3 Microtubule-Associated Protein,Microtubule Associated Proteins,Microtubule-Associated Protein 1,Microtubule-Associated Protein 2,Microtubule-Associated Protein 3,7, Microtubule-Associated Protein,Associated Protein, Microtubule,E MAP 115,Epithelial Microtubule Associate Protein, 115 kDa,MAP1 Microtubule Associated Protein,MAP2 Microtubule Associated Protein,MAP3 Microtubule Associated Protein,Microtubule Associated Protein 1,Microtubule Associated Protein 2,Microtubule Associated Protein 3,Microtubule-Associated Protein, MAP1,Microtubule-Associated Protein, MAP2,Microtubule-Associated Protein, MAP3,Protein 7, Microtubule-Associated,Protein, Microtubule Associated,Protein, Microtubule-Associated
D011494 Protein Kinases A family of enzymes that catalyze the conversion of ATP and a protein to ADP and a phosphoprotein. Protein Kinase,Kinase, Protein,Kinases, Protein
D002118 Calcium A basic element found in nearly all tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes. Coagulation Factor IV,Factor IV,Blood Coagulation Factor IV,Calcium-40,Calcium 40,Factor IV, Coagulation
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000071456 Sequestosome-1 Protein A multidomain protein that is highly conserved among multicellular organisms. It contains a ZZ-type ZINC FINGER domain, C-terminal UBIQUITIN - associated (UBA) domain, and interacts with many other signaling proteins and enzymes including, atypical PROTEIN KINASE C; TNF RECEPTOR-ASSOCIATED FACTOR 6; subunits of the mTORC1 complex, and CASPASE-8. It functions in AUTOPHAGY as a receptor for the degradation of ubiquitinated substrates, and to co-ordinate signaling in response to OXIDATIVE STRESS. EBI3-Associated Protein of 60 KDa,EBIAP Protein,Phosphotyrosine-Independent Ligand For The Lck SH2 Domain Of 62 Kda,Ubiquitin-Binding Protein p62,EBI3 Associated Protein of 60 KDa,Phosphotyrosine Independent Ligand For The Lck SH2 Domain Of 62 Kda,Sequestosome 1 Protein,Ubiquitin Binding Protein p62
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001343 Autophagy The segregation and degradation of various cytoplasmic constituents via engulfment by MULTIVESICULAR BODIES; VACUOLES; or AUTOPHAGOSOMES and their digestion by LYSOSOMES. It plays an important role in BIOLOGICAL METAMORPHOSIS and in the removal of bone by OSTEOCLASTS. Defective autophagy is associated with various diseases, including NEURODEGENERATIVE DISEASES and cancer. Autophagocytosis,ER-Phagy,Lipophagy,Nucleophagy,Reticulophagy,Ribophagy,Autophagy, Cellular,Cellular Autophagy,ER Phagy
D012694 Serine A non-essential amino acid occurring in natural form as the L-isomer. It is synthesized from GLYCINE or THREONINE. It is involved in the biosynthesis of PURINES; PYRIMIDINES; and other amino acids. L-Serine,L Serine
D014409 Tumor Necrosis Factor-alpha Serum glycoprotein produced by activated MACROPHAGES and other mammalian MONONUCLEAR LEUKOCYTES. It has necrotizing activity against tumor cell lines and increases ability to reject tumor transplants. Also known as TNF-alpha, it is only 30% homologous to TNF-beta (LYMPHOTOXIN), but they share TNF RECEPTORS. Cachectin,TNF-alpha,Tumor Necrosis Factor Ligand Superfamily Member 2,Cachectin-Tumor Necrosis Factor,TNF Superfamily, Member 2,TNFalpha,Tumor Necrosis Factor,Cachectin Tumor Necrosis Factor,Tumor Necrosis Factor alpha
D051017 Apoptosis Regulatory Proteins A large group of proteins that control APOPTOSIS. This family of proteins includes many ONCOGENE PROTEINS as well as a wide variety of classes of INTRACELLULAR SIGNALING PEPTIDES AND PROTEINS such as CASPASES. Anti-Apoptotic Protein,Anti-Apoptotic Proteins,Apoptosis Inducing Protein,Apoptosis Inhibiting Protein,Apoptosis Regulatory Protein,Pro-Apoptotic Protein,Pro-Apoptotic Proteins,Programmed Cell Death Protein,Apoptosis Inducing Proteins,Apoptosis Inhibiting Proteins,Death Factors (Apoptosis),Programmed Cell Death Proteins,Survival Factors (Apoptosis),Anti Apoptotic Protein,Anti Apoptotic Proteins,Inducing Protein, Apoptosis,Inducing Proteins, Apoptosis,Inhibiting Protein, Apoptosis,Inhibiting Proteins, Apoptosis,Pro Apoptotic Protein,Pro Apoptotic Proteins,Protein, Anti-Apoptotic,Protein, Apoptosis Inducing,Protein, Apoptosis Inhibiting,Protein, Apoptosis Regulatory,Protein, Pro-Apoptotic,Proteins, Anti-Apoptotic,Proteins, Apoptosis Inducing,Proteins, Apoptosis Inhibiting,Proteins, Pro-Apoptotic,Regulatory Protein, Apoptosis,Regulatory Proteins, Apoptosis

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