AMPK-independent induction of autophagy by cytosolic Ca super(2+) 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 super(2+) can induce autophagy by inhibition of mTOR via the CaMKK-a/b-mediated activation of AMPK. Here we demonstrate that Ca super(2+) signaling can additionally induce autophagy independently of the Ca super(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 super(2+) induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca super(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 super(2+) by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca super(2+)-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca super(2+) flux modifications. Importantly, using AMPK-a1 super(-/-)a2 super(-/-) 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-a/b 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 super(2+)-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.