title: Ca2+/calmodulin-dependent kinase (CaMK) signaling via CaMKI and AMP-activated protein kinase contributes to the regulation of WIPI-1 at the onset of autophagy.
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 Ca(2+)-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 Ca(2+) chelation by BAPTA-AM (10 and 30 μM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective Ca(2+)/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 Ca(2+) 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) α(1)/α(2)-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 Ca(2+)-dependent signaling, including CaMKI independent of AMPKα(1)/α(2). Our data also suggest that AMPKα(1)/α(2) might differentially contribute to the regulation of WIPI-1 at the onset of autophagy.
authors: Autophagy Laboratory, Interfaculty Institute for Cell Biology, Eberhard Karls University Tuebingen, Tuebingen, Germany.
authors: Simon G
authors: SG
authors: Pfisterer
authors: Mario
authors: M
authors: Mauthe
authors: Patrice
authors: P
authors: Codogno
authors: Tassula
authors: T
authors: Proikas-Cezanne
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001343:Autophagy / Q000502:physiology*; D000071183:Autophagy-Related Proteins; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; C522812:WIPI1 protein, human; C526181:PRKAA2 protein, human; C517596:CAMK1 protein, human; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human