title: A comprehensive siRNA screen for kinases that suppress macroautophagy in optimal growth conditions.
abstract: Macroautophagy is a catabolic process that maintains cellular homeostasis and protects cells against various external stresses including starvation. Except for the identification of the Akt-mTORC1 pathway as a major negative regulator, little is known about signaling networks that control macroautophagy under optimal growth conditions. Therefore, we screened a human kinome siRNA library for siRNAs that increase the number of autophagosomes in normally growing MCF-7 human breast carcinoma cells, and identified 10 kinases as regulators of constitutive macroautophagy. Further analysis of these kinases with respect to the autophagic flux, kinase signaling and endolysosomal function identified WNK2 as a positive regulator of autophagosome maturation and nine others as macroautophagy inhibitors. The depletion of MK2, PACSIN1, DAPK2, CDKL3 and SCYL1 functioned upstream of Akt-mTORC1 pathway, whereas CSNK1A1, BUB1, PKLR and NEK4 suppressed autophagosome formation downstream or independent of mTORC1. Importantly, all identified kinases except for BUB1 regulated macroautophagy also in immortalized MCF-10A breast epithelial cells. The kinases identified here shed light to the complex regulation of macroautophagy and open new possibilities for its pharmacological manipulation.
authors.affiliation: Apoptosis Department and Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark.
authors.forename: Piotr
authors.initials: P
authors.lastname: Szyniarowski
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Corcelle-Termeau
authors.forename: Thomas
authors.initials: T
authors.lastname: Farkas
authors.forename: Maria
authors.initials: M
authors.lastname: Høyer-Hansen
authors.forename: Jesper
authors.initials: J
authors.lastname: Nylandsted
authors.forename: Tuula
authors.initials: T
authors.lastname: Kallunki
authors.forename: Marja
authors.initials: M
authors.lastname: Jäättelä
mesh_terms: D001343:Autophagy; D001940:Breast / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D046912:Multiprotein Complexes; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases / Q000378:metabolism*; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases
chemical_list: D000076222:Mechanistic Target of Rapamycin Complex 1; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases; D011506:Proteins; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D058570:TOR Serine-Threonine Kinases; C477012:MAP1LC3A protein, human