AMPK-independent induction of autophagy by cytosolic Ca2+ increase

Cell Signal. 2010 Jun;22(6):914-25. doi: 10.1016/j.cellsig.2010.01.015. Epub 2010 Jan 28.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy*
  • Calcium / metabolism*
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase / metabolism
  • Carrier Proteins / analysis
  • Cell Line
  • Chelating Agents / pharmacology
  • Cytosol / metabolism*
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / pharmacology
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Mice
  • Microtubule-Associated Proteins / analysis
  • Microtubule-Associated Proteins / metabolism
  • Phagosomes / drug effects
  • Phosphatidylinositols / metabolism
  • Protein Kinases / metabolism*
  • Protein-Serine-Threonine Kinases / metabolism
  • TOR Serine-Threonine Kinases
  • Thapsigargin / pharmacology

Substances

  • Carrier Proteins
  • Chelating Agents
  • Intracellular Signaling Peptides and Proteins
  • Microtubule-Associated Proteins
  • Phosphatidylinositols
  • phosphoinositide 3-phosphate
  • 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester
  • Egtazic Acid
  • Thapsigargin
  • Protein Kinases
  • AMP-activated protein kinase kinase
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • mTOR protein, mouse
  • Protein-Serine-Threonine Kinases
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase
  • Calcium