Abstract
Autophagy is an intracellular degradation process essential for adaptation to metabolic stress. DAPK2 is a calmodulin-regulated protein kinase, which has been implicated in autophagy regulation, though the mechanism is unclear. Here, we show that the central metabolic sensor, AMPK, phosphorylates DAPK2 at a critical site in the protein structure, between the catalytic and the calmodulin-binding domains. This phosphorylation activates DAPK2 by functionally mimicking calmodulin binding and mitigating an inhibitory autophosphorylation, providing a novel, alternative mechanism for DAPK2 activation during metabolic stress. In addition, we show that DAPK2 phosphorylates the core autophagic machinery protein, Beclin-1, leading to dissociation of its inhibitor, Bcl-XL. Importantly, phosphorylation of DAPK2 by AMPK enhances DAPK2’s ability to phosphorylate Beclin-1, and depletion of DAPK2 reduces autophagy in response to AMPK activation. Our study reveals a unique calmodulin-independent mechanism for DAPK2 activation, critical to its function as a novel downstream effector of AMPK in autophagy.
Highlights
Autophagy is an intracellular degradation process essential for adaptation to metabolic stress
We show that AMPK phosphorylates Death-associated protein kinase 2 (DAPK2) on Ser[289] in cell cultures exposed to metabolic stress and in muscle tissue of fasted mice
We show that DAPK2 phosphorylates the core autophagic machinery protein, Beclin-1, on Thr[119] located in its BH3 domain, causing it to dissociate from its inhibitor Bcl-XL
Summary
Autophagy is an intracellular degradation process essential for adaptation to metabolic stress. We show that the central metabolic sensor, AMPK, phosphorylates DAPK2 at a critical site in the protein structure, between the catalytic and the calmodulin-binding domains. This phosphorylation activates DAPK2 by functionally mimicking calmodulin binding and mitigating an inhibitory autophosphorylation, providing a novel, alternative mechanism for DAPK2 activation during metabolic stress. The founder member of the family is DAPK1, a large multi-domain Ca2+/CaM-regulated Ser/Thr kinase implicated in a wide range of biological activities Subsequent knock-down experiments confirmed the involvement of DAPK2 in regulating autophagy in human cell lines[19] and preadipocytes[20]
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