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

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.

Automated summary

MLKL plays a key role in cell death and endosomal trafficking, with CAMK2-mediated phosphorylation promoting autophagy during short-term starvation. This pathway protects cells from starvation-induced death and is independent of RIPK3, suggesting new roles for the pseudokinase in cell survival and metabolism.