Regulation of GSK3 cellular location by FRAT modulates mTORC1-dependent cell growth and sensitivity to rapamycin Long Hea,b,c,1, Dennis Liang Feia, Michal J. Nagieca,b, Anders P. Mutveia,b, Andreas Lamprakisa,b, Bo Yeon Kimc, and John Blenisa,b,1 aMeyer Cancer Center, Weill Cornell Medical College, New York, NY 10021; bDepartment of Pharmacology, Weill Cornell Medical College, New York, NY 10021; and cWorld Class Institute, Korea Research Institute of Bioscience and Biotechnology, 363-883 Ochang, Korea Edited by Michael N. Hall, University of Basel, Basel, Switzerland, and approved August 5, 2019 (received for review February 10, 2019) The mTORC1 pathway regulates cell growth and proliferation by stimulation by multiple growth factors (3). This suppression is properly coupling critical processes such as gene expression, protein predominantly achieved via phosphorylation at Ser21 or Ser9, translation, and metabolism to the availability of growth factors and respectively, on GSK3α/β by multiple kinases (5). For example, hormones, nutrients, cellular energetics, oxygen status, and cell inhibitory phosphorylation of GSK3 was shown to be regulated by stress. Although multiple cytoplasmic substrates of mTORC1 have mTORC1/S6K1 and contribute to mTORC1-mediated suppression been identified, how mTORC1 signals within the nucleus remains of GSK3 activity (6). Interestingly, high levels of inhibitory GSK3 incompletely understood. Here, we report a mechanism by which phosphorylation or GSK3 inhibitors were reported to confer mTORC1 modulates the phosphorylation of multiple nuclear events. rapamycin resistance in certain cell types (7, 8). GSK3 also phos- We observed a significant nuclear enrichment of GSK3 when mTORC1 phorylates and inhibits multiple nuclear proteins upon pharmaco- was suppressed, which promotes phosphorylation of several proteins logical mTORC1 suppression, such as TIP60, c-Myc, and FOXK1 such as GTF2F1 and FOXK1. Importantly, nuclear localization of GSK3 (6, 9, 10). Therefore, one prediction is that, upon rapamycin is sufficient to suppress cell proliferation. Additionally, expression of treatment, GSK3 inhibitory phosphorylation is lost, resulting in inhibition of proliferation. However, it has been reported that a nuclear exporter of GSK3, FRAT, restricts the nuclear localization CELL BIOLOGY GSK3 phosphorylation was not significantly suppressed by of GSK3, represses nuclear protein phosphorylation, and prevents rapamycin treatment in multiple cell lines (8). Additionally, we have rapamycin-induced cytostasis. Finally, we observe a correlation be- observed that GSK3-dependent regulation of FOXK1 phosphory- tween rapamycin resistance and FRAT expression in multiple-cancer lation occurred under conditions where GSK3 phosphorylation cell lines. Resistance to Food and Drug Administration (FDA)- was not significantly affected upon mTORC1 suppression, suggesting approved rapamycin analogs (rapalogs) is observed in many tumor a possible phosphorylation-independent mechanism for regu- settings, but the underling mechanisms remain incompletely under- lation of GSK3 by mTORC1 signaling (9). Thus, the relationship stood. Given that FRAT expression levels are frequently elevated in between mTORC1, GSK3, and rapamycin resistance remains various cancers, our observations provide a potential biomarker and unclear. strategy for overcoming rapamycin resistance. Here we report that the nuclear−cytoplasmic localization of GSK3 is regulated by mTORC1 signaling, which subsequently mTOR | GSK3 | FRAT | Resistance contributes to multiple nuclear protein phosphorylation events. The mTORC1 suppression leads to accumulation of GSK3 in the he protein kinase complex mechanistic target of rapamycin nucleus, thereby promoting the phosphorylation of GSK3 targets Tcomplex 1 (mTORC1) is a central regulator of mammalian cell growth and proliferation that is dysregulated in various Significance cancer types, including colorectal, breast, lung, renal cell carci- noma, glioblastoma, and prostate cancers (1). Several rapalogs The clinical effectiveness of FDA-approved anticancer rapalogs is (rapamycin and its derivatives) have been developed and Food limited because resistance is observed in multiple-tumor set- and Drug Administration (FDA)-approved for cancer treatment tings. In order to improve the efficacy of rapalogs, there is a (2). However, the impact of rapalogs in the clinic has been limited, need to improve our ability to predict and overcome rapamycin as resistance to rapalogs is frequently observed upon long-term resistance. Here we report that high FRAT1/2 expression confers treatment. In order to improve anticancer efficacy of rapalogs, rapamycin resistance by excluding GSK3 from the nucleus in there is a need to improve our understanding of these resistance multiple-cancer cell lines. FRAT1 expression is frequently high in mechanisms. As a central signaling cascade, once activated, mTORC1 multiple cancers, and our observations provide evidence for promotes multiple anabolic processes by phosphorylating a num- using FRAT1/2 expression as a biomarker for rapamycin sensi- ber of downstream substrates, including S6K1, 4EBPs, Grb10, tivity. Moreover, our findings reveal a major mechanism for how Lipin1, and others (1). Changes in mTORC1 signaling are also mTORC1 regulates nuclear protein phosphorylation and signal- understood to influence several biological processes that are ing, and uncover a potential therapeutic approach for targeting spatially restricted to the nucleus, including gene transcription, mTORC1-driven cancers. messenger RNA (mRNA) splicing, and chromosome remodel- ing. However, despite the significant advances made in un- Author contributions: L.H., B.Y.K., and J.B. designed research; L.H., D.L.F., and A.L. per- derstanding the mTORC1 pathway in the past 2 decades, how formed research; A.P.M. analyzed data; and L.H., M.J.N., and J.B. wrote the paper. mTORC1 signaling controls nuclear events is still not fully de- The authors declare no conflict of interest. fined, and whether nuclear signaling contributes to rapamycin This article is a PNAS Direct Submission. resistance observed during chronic treatment is unknown. Published under the PNAS license. GSK3 is known to phosphorylate a large number of substrates 1To whom correspondence may be addressed. Email: [email protected] or important in regulating cell growth and metabolism (3). Inap- [email protected]. propriate regulation of GSK3 is linked to several disorders, in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cluding cancer (3, 4). In contrast to mTORC1, GSK3α/β have 1073/pnas.1902397116/-/DCSupplemental. relatively high activity in resting cells, which is suppressed upon www.pnas.org/cgi/doi/10.1073/pnas.1902397116 PNAS Latest Articles | 1of7 Downloaded by guest on October 1, 2021 such as FOXK1 and GTF2F1. Critically, we found the GSK3 Results interacting proteins, frequently rearranged in advanced T lymphomas Nuclear Enrichment of GSK3 Promotes Protein Phosphorylation upon 1 and 2 (FRAT1 and 2), which promote GSK3 nuclear export, pre- mTORC1 Suppression. The mTORC1 and downstream effectors vent rapamycin-induced nuclear accumulation of GSK3 and phos- such as ULK1/2, S6 protein kinases (S6K1/2), and SRPK2 reg- phorylation of its targets. Given these observations, we asked whether ulate the phosphorylation of a variety of proteins that affect mTORC1-GSK3 nuclear signaling was a major contributor to cell multiple cellular events associated with metabolism, mRNA proliferation and whether failure to promote GSK3 nuclear signaling processing and translation, survival signaling, autophagy, and cell might contribute to rapamycin resistance. In support of this possibility, growth (1, 11). An important observation made upon analysis of we observed that ectopic expression of GSK3 that is forced to locate the mTORC1 phosphoproteome revealed that, in addition to the in the nucleus (NLS-GSK3) promotes phosphorylation of proteins promotion of phosphorylation via mTORC1 signaling, the such as FOXK1 and GTF2F1, and, importantly, suppresses cancer phosphorylation of ∼100 proteins appeared to be suppressed by cell proliferation. We also observed that overexpression FRAT1/2 in mTORC1 (12). To investigate this unexpected role of mTORC1 rapamycin-sensitive cancer cells promotes resistance to the cytostatic in suppressing protein phosphorylation, we characterized phos- effects of rapamycin, consistent with the acquisition of drug resistance. phorylation of a transcription factor FOXK1, and demonstrated Consistently, cells with high FRAT1/2 expression are relatively in- that mTORC1 suppresses FOXK1 phosphorylation in a GSK3- sensitive to rapamycin treatment, and down-regulation of FRAT1/2 dependent manner (9). Furthermore, many of the phosphopro- with RNA interference (RNAi) resensitizes cells to rapamycin. Our teins identified in this analysis are also nuclear resident proteins SI Appendix observations in xenograft models further support the conclusion that with potential, conserved GSK3 sites ( , Table S1). To elevated expression of FRAT1/2, and suppression of GSK3 nuclear determine whether there are additional proteins whose phos- flux, are potential mechanisms by which cancer cells acquire rapa- phorylation is suppressed by mTORC1, we utilized available mycin resistance. Collectively, our findings demonstrate that moni- reagents to monitor the phosphorylation of another nuclear toring FRAT1/2 expression levels and/or GSK3 localization