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

Szyniarowski, Piotr; Corcelle-Termeau, Elisabeth; Farkas, Thomas; Høyer-Hansen, Maria; Nylandsted, Jesper; Kallunki, Tuula; Jäättelä, Marja

doi:10.4161/auto.7.8.15770

Cited by 76 publications

(74 citation statements)

References 49 publications

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“…During a recent study of casein kinase 1 (CK1) in the regulation of cancer cell growth (19), we noted a role for CK1α in the modulation of oncogenic RASinduced autophagic flux. This observation is consistent with a recent kinome RNAi screen that identified CK1 isoforms as constitutive autophagy-regulating kinases in human breast cancer cells (20). The CK1 family of ubiquitously expressed serine/threonine kinases consists of six human isoforms (α, δ, ε, γ1, γ2, and γ3) that are evolutionary conserved within eukaryotes (21,22).…”

Section: Introductionsupporting

confidence: 77%

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Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers

Cheong¹,

Zhang²,

Chua³

et al. 2015

J. Clin. Invest.

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Section: Introductionsupporting

confidence: 77%

“…A recent kinome RNAi screen scored CK1α as a significant basal autophagy-regulating kinase in MCF-7 human breast cancer cells (20). In that study, the authors attributed the enhancement (D4476:CQ; Figure 7A).…”

Section: Discussionmentioning

confidence: 97%

Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers

Cheong¹,

Zhang²,

Chua³

et al. 2015

J. Clin. Invest.

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“…autophagosome-lysosome fusion and autolysosomal degradation, are less prone for inhibition simply because kinase signaling plays a less prominent role for these processes or because of great redundancy in signaling pathways. Emphasizing that maturation is potentially difficult to target via kinase inhibition, we recently published a screen of a siRNA library targeting the human kinome (726 known and putative kinases) for siRNAs that induce an accumulation of LC3-positive autophagosomes in MCF7 cells, and WNK lysine-deficient protein kinase 2 (WNK2) was the sole kinase identified as a positive regulator of maturation in this screen (34).…”

Section: Discussionmentioning

confidence: 99%

Identification of Small Molecule Inhibitors of Phosphatidylinositol 3-Kinase and Autophagy

Farkas

Daugaard

Jäättelä

2011

Journal of Biological Chemistry

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Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered by the lack of specific small molecule inhibitors. Thus, we screened two small molecule kinase inhibitor libraries for inhibitors of rapamycin-induced autophagic flux. The three most potent inhibitors identified conferred profound inhibition of autophagic flux by inhibiting the formation of autophagosomes. Notably, the autophagy inhibitory effects of all three compounds were independent of their established kinase targets, i.e. ataxia telangiectasia mutated for KU55933, protein kinase C for Gö6976, and Janus kinase 3 for Jak3 inhibitor VI. Instead, we identified phosphatidylinositol 3-kinase (PtdIns3K) as a direct target of KU55933 and Gö6976. Importantly, and in contrast to the currently available inhibitors of autophagosome formation (e.g. 3-methyladenine), none of the three compounds inhibited the cell survival promoting class I phosphoinositide 3-kinase-Akt signaling at the concentrations required for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new possibilities for the experimental study of autophagy and can form a basis for the development of clinically relevant autophagy inhibitors.Autophagy is an intracellular degradative process by which cells recycle macromolecules and organelles (1-4). In this process, cellular material is sequestered in double membrane vesicles termed autophagosomes that fuse with lysosomes to form autolysosomes, in which the cargo is exposed to acidic hydrolases. Autophagy is essential for energy homeostasis and removal of damaged organelles and protein complexes during various kinds of stresses, such as starvation, growth factor deprivation, hypoxia, and DNA damage. It is also involved in physiological processes like development, immunity, and aging as well as in various diseases including neurodegenerative disorders and cancer. Whereas autophagy clearly has a beneficial effect in preventing many degenerative disorders, its role in cancer is more complex. It can function as a tumor suppressor mechanism, and yet it can also promote tumor growth by protecting cancer cells against the hostile tumor environment and antineoplastic drugs (5, 6).The mammalian target of rapamycin complex 1 (mTORC1) 3 serine/threonine kinase integrates information on cell metabolic, growth, and stress status to regulate biosynthetic pathways and autophagy (7,8). It activates biosynthetic pathways and inhibits autophagy in response to various growth factors via MAPK/ERK and class I phosphoinositide 3-kinase (PI3K)/Aktdependent pathways. On the other hand, when the ene...

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“…In other contexts, a robust loss of SQSTM1 does not correlate with increased autophagic flux as assessed by a luciferase-based measure of flux. 166 Thus, appropriate positive and negative controls, and assessment of SQSTM1 mRNA levels, may be needed prior to the use of SQSTM1 as a flux indicator in a particular cellular context.…”

mentioning

confidence: 99%

“…This method has been successfully used to identify positive and negative regulators of autophagy from cells treated with microRNA, siRNA and small molecule libraries. [163][164][165][166] Cautionary notes: The main caveat regarding the measurement of LC3-IIs/LC3-I is that this method has only been tested in isolated rat hepatocytes and H4-II-E cells. Thus, it is not yet known whether it is generally applicable to other cell types, and a soluble form of LC3-II (i.e., LC3-IIs) is not observed in many standard cell types including HeLa, HEK 293 and PC12.…”

mentioning

confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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

Cited by 76 publications

References 49 publications

Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers

Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers

Identification of Small Molecule Inhibitors of Phosphatidylinositol 3-Kinase and Autophagy

Guidelines for the use and interpretation of assays for monitoring autophagy

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