Editors' ChoiceCell Biology

How to Stop Responding to Stressful Conditions

Science Signaling  20 Aug 2013:
Vol. 6, Issue 289, pp. ec198
DOI: 10.1126/scisignal.2004645

Mechanistic target of rapamycin (mTOR) is a kinase that assembles with the regulatory component raptor to form mTOR complex 1 (mTORC1) or the regulatory component rictor to form mTORC2. mTORC1, which couples metabolic and stress signals with pathways that mediate cellular growth and proliferation, is both stimulated and inhibited by oxidative stress. Thedieck et al. sought to characterize how mTORC1 is inhibited by oxidative stress. Mass spectrometry analysis of immunoprecipitates of endogenous mTOR, raptor, and rictor from HeLa cells identified astrin as a binding partner for raptor but not for mTOR or rictor. Astrin promotes mitotic progression, and increased expression of the mRNA encoding astrin correlates with a poor prognosis in breast and lung cancer. Treatment of cells with the mTORC1 inhibitor rapamycin did not affect the binding of astrin to raptor, whereas siRNA-mediated knockdown of astrin increased the association of mTOR with raptor. Phosphorylation and activation of the mTORC1 target S6K1 was increased by knockdown of astrin in unsynchronized HeLa cells but not cells that were arrested in G2/M, which led the authors to suggest that astrin inhibited mTORC1 independently of its role in mitosis. In insulin-treated cells, knockdown of astrin increased the phosphorylation of S6K1, an effect that was blocked by rapamycin and the nonselective mTORC inhibitor PP242. Furthermore, knockdown of astrin enhanced the phosphorylation of the S6K1 target IRS1 (insulin receptor substrate 1) and accelerated that of S6K1 and PRAS40, another mTORC1 substrate. Astrin tagged with green fluorescent protein (GFP) colocalized with the stress granule component p54. In cells treated with arsenite to induce the formation of stress granules, GFP-astrin colocalized with the stress granule marker G3BP1 and HA-tagged raptor. Compared with unstressed cells, the interaction of astrin with raptor or G3BP1 was increased, whereas raptor association with mTOR was decreased in arsenite- or hydrogen peroxide–treated cells, an effect that was reduced by astrin knockdown in arsenite-treated cells. Arsenite increased the phosphorylation of S6K1, an effect that was increased by astrin knockdown and that was attenuated by rapamycin or knockdown of raptor. Astrin knockdown increased the phosphorylation of S6K1 in response to various treatments that induce the formation of stress granules, suggesting that astrin limits mTORC1 activity during stress. mTORC1 is active at lysosomes, and astrin knockdown or arsenite treatment decreased the association of raptor with the lysosomal marker Lamp2. In arsenite-treated cells, the ability of astrin to limit mTORC1 activity was reduced by cycloheximide treatment, which prevents stress granule assembly. Tumor cells must cope with redox stress, a condition that can induce apoptosis, and increased mTORC1 activity can promote apoptosis. In hydrogen peroxide–treated MCF-7 breast cancer cells, astrin knockdown enhanced apoptosis, an effect that was reversed by mTORC1 inhibition, and accelerated the onset of apoptotic membrane blebbing, an effect that was reduced by cycloheximide treatment. Thus, astrin limits mTORC1 activation in response to stress by preventing the association of raptor and mTOR; astrin could be targeted to sensitize cancer cells to apoptosis.

K. Thedieck, B. Holzwarth, M. T. Prentzell, C. Boehlke, K. Kläsener, S. Ruf, A. G. Sonntag, L. Maerz, S.-N. Grellscheid, E. Kremmer, R. Nitschke, E. W. Kuehn, J. W. Jonker, A. K. Groen, M. Reth, M. N. Hall, R. Baumeister, Inhibition of mTORC1 by astrin and stress granules prevents apoptosis in cancer cells. Cell 154, 859–874 (2013). [Abstract]