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Molecular Pharmacology

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Research ArticleArticle

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

Simon G. Pfisterer, Mario Mauthe, Patrice Codogno and Tassula Proikas-Cezanne
Molecular Pharmacology December 2011, 80 (6) 1066-1075; DOI: https://doi.org/10.1124/mol.111.071761
Simon G. Pfisterer
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Mario Mauthe
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Patrice Codogno
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Tassula Proikas-Cezanne
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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

  • ↵Embedded Image 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.

  • 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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Molecular Pharmacology: 80 (6)
Molecular Pharmacology
Vol. 80, Issue 6
1 Dec 2011
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Research ArticleArticle

CaMKs Contribute to WIPI-1 Regulation at Autophagy Onset

Simon G. Pfisterer, Mario Mauthe, Patrice Codogno and Tassula Proikas-Cezanne
Molecular Pharmacology December 1, 2011, 80 (6) 1066-1075; DOI: https://doi.org/10.1124/mol.111.071761

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Research ArticleArticle

CaMKs Contribute to WIPI-1 Regulation at Autophagy Onset

Simon G. Pfisterer, Mario Mauthe, Patrice Codogno and Tassula Proikas-Cezanne
Molecular Pharmacology December 1, 2011, 80 (6) 1066-1075; DOI: https://doi.org/10.1124/mol.111.071761
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