AMPK-independent induction of autophagy by cytosolic Ca2+ increase.
Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca(2+) can induce autophagy by inhibition of mTOR via the CaMKK-alpha/beta-mediated activation of AMPK. Here we demonstrate that Ca(2+) signaling can additionally induce autophagy independently of the Ca(2+)-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca(2+) induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca(2+)-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca(2+) by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca(2+)-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca(2+) flux modifications. Importantly, using AMPK-alpha1(-/-)alpha2(-/-) MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-alpha/beta inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca(2+)-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.
Grotemeier
Antje
A
Department of Internal Medicine I, University Clinic of Tübingen, Germany.
Alers
Sebastian
S
Pfisterer
Simon G
SG
Paasch
Florian
F
Daubrawa
Merle
M
Dieterle
Alexandra
A
Viollet
Benoit
B
Wesselborg
Sebastian
S
Proikas-Cezanne
Tassula
T
Stork
Björn
B
D000091162:AMP-Activated Protein Kinase Kinases; D000818:Animals; D001343:Autophagy; D002118:Calcium / Q000378:metabolism*; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects; D010716:Phosphatidylinositols / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D019284:Thapsigargin / Q000494:pharmacology
D002352:Carrier Proteins; D002614:Chelating Agents; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D010716:Phosphatidylinositols; C483312:phosphoinositide 3-phosphate; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019284:Thapsigargin; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D000091162:AMP-Activated Protein Kinase Kinases; D002118:Calcium