Abstract
Autophagy is initiated by multimembrane vesicle (autophagosome) formation upon mammalian target of rapamycin inhibition and phosphatidylinositol 3-phosphate [PtdIns(3)P] generation. Upstream of microtubule-associated protein 1 light chain 3 (LC3), WD-repeat proteins interacting with phosphoinositides (WIPI proteins) specifically bind PtdIns(3)P at forming autophagosomal membranes and become membrane-bound proteins of generated autophagosomes. Here, we applied automated high-throughput WIPI-1 puncta analysis, paralleled with LC3 lipidation assays, to investigate Ca2+-mediated autophagy modulation. We imposed cellular stress by starvation or administration of etoposide (0.5–50 μM), sorafenib (1–40 μM), staurosporine (20–500 nM), or thapsigargin (20–500 nM) (1, 2, or 3 h) and measured the formation of WIPI-1 positive autophagosomal membranes. Automated analysis of up to 5000 individual cells/treatment demonstrated that Ca2+ chelation by BAPTA-AM (10 and 30 μM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective Ca2+/calmodulin-dependent kinase kinase (CaMKK) α/β and calmodulin-dependent kinase (CaMK) I/II/IV inhibitors 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609; 10–30 μg/ml) and 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93; 1–10 μM), respectively, significantly reduced starvation-induced autophagosomal membrane formation, suggesting that Ca2+ mobilization upon autophagy induction involves CaMKI/IV. By small interefering RNA (siRNA)-mediated down-regulation of CaMKI or CaMKIV, we demonstrate that CaMKI contributes to stimulation of WIPI-1. In line, WIPI-1 positive autophagosomal membranes were formed in AMP-activated protein kinase (AMPK) α1/α2-deficient mouse embryonic fibroblasts upon nutrient starvation, whereas basal autophagy was prominently reduced. However, transient down-regulation of AMPK by siRNA resulted in an increased basal level of both WIPI-1 puncta and LC3 lipidation, and nutrient-starvation induced autophagy was sensitive to STO-609/KN-93. Our data provide evidence that pharmacological compound-modulated and starvation-induced autophagy involves Ca2+-dependent signaling, including CaMKI independent of AMPKα1/α2. Our data also suggest that AMPKα1/α2 might differentially contribute to the regulation of WIPI-1 at the onset of autophagy.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This study was supported by the Deutsche Forschungsgemeinschaft [Grants SFB773, A03] and the Bundesministerium für Bildung und Forschung [Grant FKZ 031 3816B].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.071761.
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ABBREVIATIONS:
- PtdIns3KC3
- phospatidylinositol-3 kinase class III
- TORC1
- mTOR complex 1
- LC3
- microtubule-associated protein 1 light chain 3
- PtdIns(3)P
- phosphatidylinositol 3-phosphate
- WIPI
- WD-repeat protein interacting with phosphoinositides
- mTOR
- mammalian target of rapamycin
- CaMK
- calmodulin-dependent kinase
- CaMKK
- calmodulin-dependent kinase kinase
- AMPK
- AMP-activated protein kinase
- STO-609
- 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate
- KN-93
- 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine
- Baf
- bafilomycin
- DAPI
- 4,6-diamidino-2-phenylindole
- BAPTA-AM
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester
- DMEM
- Dulbecco's modified Eagle's medium
- FCS
- fetal calf serum
- GFP
- green fluorescent protein
- MEF
- mouse embryonic fibroblast
- SF
- sorafenib
- SP
- staurosporine
- TG
- thapsigargin
- EP
- etoposide
- NF
- nutrient-free medium lacking amino acids and serum
- CM
- control medium
- WM
- wortmannin
- WT
- wild-type
- LY294002
- 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one.
- Received February 14, 2011.
- Accepted September 6, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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