TY - JOUR T1 - Ca<sup>2+</sup>/Calmodulin-dependent Kinase Signaling via CaMKI and AMPK Contributes to the Regulation of WIPI-1 at the Onset of Autophagy JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.111.071761 SP - mol.111.071761 AU - Simon G Pfisterer AU - Mario Mauthe AU - Patrice Codogno AU - Tassula Proikas-Cezanne Y1 - 2011/09/06 UR - http://molpharm.aspetjournals.org/content/early/2011/09/06/mol.111.071761.abstract N2 - Autophagy is initiated by multi-membrane vesicle (autophagosome) formation upon mTOR inhibition and phosphatidylinositol 3-phosphate (PtdIns(3)P) generation. Upstream of LC3, 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, etoposide (0.5 - 50 µM), sorafenib (1 - 40 µM), staurosporine (20 - 500 nM) or thapsigargin (20 – 500 nM) administration (1, 2 or 3 hrs) 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, 30 µM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective CaMKKα/β and CaMKI/II/IV inhibitors, respectively STO-609 (10 - 30 µg/ml) and KN-93 (1 – 10 µM), significantly reduced starvation-induced autophagosomal membrane formation, suggesting that Ca2+ mobilization upon autophagy induction involves CaMKI/IV. By siRNA-mediated downregulation of CaMKI or CaMKIV, we demonstrate that CaMKI contributes to stimulation of WIPI-1. In line, WIPI-1 positive autophagosomal membranes were formed in AMPKα1/α2-deficient MEFs upon nutrient starvation, while basal autophagy was prominently reduced. However, transient downregulation 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. ER -