TY - JOUR

T1 - A comprehensive siRNA screen for kinases that suppress macroautophagy in optimal growth conditions

AU - Szyniarowski, Piotr

AU - Corcelle-Termeau, Elisabeth

AU - Farkas, Thomas

AU - Høyer-Hansen, Maria

AU - Nylandsted, Jesper

AU - Kallunki, Tuula

AU - Jaattela, Marja

PY - 2011

Y1 - 2011

N2 - 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.

AB - 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.

KW - Autophagy

KW - Breast

KW - Cell Line, Tumor

KW - Cell Proliferation

KW - Endocytosis

KW - Epithelial Cells

KW - Female

KW - Genetic Testing

KW - Humans

KW - Lysosomes

KW - Microtubule-Associated Proteins

KW - Phosphatidylinositol 3-Kinases

KW - Phosphotransferases

KW - Protein Transport

KW - Proteins

KW - Proto-Oncogene Proteins c-akt

KW - RNA, Small Interfering

KW - Signal Transduction

M3 - Journal article

C2 - 21508686

VL - 7

SP - 892

EP - 903

JO - Autophagy

JF - Autophagy

SN - 1554-8627

IS - 8

ER -