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Phosphorylation of DEPDC5, a Component of the GATOR1 Complex, Releases Inhibition of Mtorc1 and Promotes Tumor Growth

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Phosphorylation of DEPDC5, a Component of the GATOR1 Complex, Releases Inhibition of Mtorc1 and Promotes Tumor Growth Phosphorylation of DEPDC5, a component of the GATOR1 complex, releases inhibition of mTORC1 and promotes tumor growth Sathish K. R. Padia, Neha Singha, Jeremiah J. Bearssb, Virginie Oliveb, Jin H. Songc, Marina Cardó-Vilad, Andrew S. Krafta,b,1, and Koichi Okumurae,1 aUniversity of Arizona Cancer Center, The University of Arizona, Tucson, AZ 85724; bDepartment of Medicine, The University of Arizona, Tucson, AZ 85724; cDepartment of Cellular and Molecular Medicine, The University of Arizona, Tucson, AZ 85724; dDepartment of Otolaryngology, The University of Arizona, Tucson, AZ 85724; and eDepartment of Physiology, The University of Arizona, Tucson, AZ 85724 Edited by Michael N. Hall, University of Basel, Basel, Switzerland, and approved August 28, 2019 (received for review March 20, 2019) The Pim and AKT serine/threonine protein kinases are implicated amino acids is regulated by a cascade of protein complexes that as drivers of cancer. Their regulation of tumor growth is closely tied function by modulating the activity of the Rag GTPases, RagA/B to the ability of these enzymes to mainly stimulate protein synthesis and RagC/D (10, 12, 13). The activity of the Rag GTPases is by activating mTORC1 (mammalian target of rapamycin complex 1) repressed by the GATOR1 complex proteins, DEPDC5, NPRL2, signaling, although the exact mechanism is not completely un- and NPRL3, based on GATOR1’s GTPase-activating protein derstood. mTORC1 activity is normally suppressed by amino acid (GAP) activity (14, 15). Since the Rheb GTPase activity is highly starvation through a cascade of multiple regulatory protein com- regulated in tumors by TSC2 phosphorylation (16, 17), we hy- plexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity pothesized that a similar mechanism could control the GATOR1 of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP complex and thus Rag GTPase and mTORC1 activity. domain containing protein 5), a component of GATOR1 complex, Here we describe the regulation of GATOR1 complex by the contains Pim and AKT protein kinase phosphorylation consensus phosphorylation of DEPDC5 mediated by the Pim and AKT sequences. DEPDC5 phosphorylation by Pim and AKT kinases was protein kinases. Deleting DEPDC5 or mutating specific phos- CELL BIOLOGY confirmed in cancer cells through the use of phospho-specific anti- phorylation sites within the protein partially blocks the antitumor bodies and transfection of phospho-inactive DEPDC5 mutants. Con- activity of small molecule inhibitors used clinically to inhibit Pim sistent with these findings, during amino acid starvation the and AKT protein kinases (18, 19). These findings shed light on a elevated expression of Pim1 overcame the amino acid inhibitory phosphorylation-dependent regulatory mechanism targeting the protein cascade and activated mTORC1. In contrast, the knockout Pim1/AKT-GATOR1-mTORC1 signaling cascade that is a driver of DEPDC5 partially blocked the ability of small molecule inhibitors of cancer cell proliferation. against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. Results In animal experiments knocking in a glutamic acid (S1530E) in Pim Kinases Regulate the Amino Acid-Sensitive mTORC1 Pathway. DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Upon amino acid starvation, the GATOR1 protein complex Our results indicate a phosphorylation-dependent regulatory mech- is recruited to the lysosome by the interaction of KICSTOR anism targeting DEPDC5 through which Pim1 and AKT act as up- components after the dissociation of the GATOR2, and this stream effectors of mTORC1 to facilitate proliferation and survival of cancer cells. Significance Pim kinase | DEPDC5 | GATOR1 | AKT kinase | mTORC1 The mTORC1 (mammalian target of rapamycin complex 1) pathway plays a critical role in driving cancer growth. We have he Pim (proviral integration site for Moloney murine leu- identified a phosphorylation-dependent mechanism that con- Tkemia virus) serine/threonine protein kinases have been im- trols mTORC1 activity in which Pim and AKT kinases, 2 enzymes plicated as a driver of both triple negative breast cancer (TNBC) with increased activity in cancer phosphorylate DEPDC5, a and advanced prostate cancer (1–3). Regulation of tumor growth member of the GATOR1 complex that senses cellular amino by Pim has been closely tied with the ability of this kinase to acid levels. The critical nature of this substrate to the activity of stimulate protein synthesis by activating mTORC1 (mammalian these protein kinases is demonstrated by the fact that deletion target of rapamycin complex 1) signaling (4, 5), but the mechanism or mutation of DEPDC5 partially blocks the ability of Pim and by which Pim regulates mTORC1 signaling is unknown. The in- Pim plus AKT inhibitors to suppress tumor cell growth. Thus, teraction between Pim and AKT kinase pathways has been well protein kinases regulate the amino acid sensing cascade to established and plays an important role in tumorigenesis, as control mTORC1 activity and tumor cell growth. demonstrated by the observation that PI3K/AKT inhibition in- Author contributions: S.K.R.P., A.S.K., and K.O. designed research; S.K.R.P., N.S., and K.O. creases Pim kinase levels thus sustaining mTORC1 activity (6, 7). performed research; S.K.R.P., N.S., J.J.B., V.O., J.H.S., M.C.-V., and K.O. contributed new It has also been shown that tumor resistance to a PI3K/AKT in- reagents/analytic tools; S.K.R.P., N.S., A.S.K., and K.O. analyzed data; and S.K.R.P., A.S.K., hibitor treatment in human breast cancer can be overcome by Pim and K.O. wrote the paper. inhibitor therapy (8), suggesting that Pim and AKT have an The authors declare no conflict of interest. overlapping mechanisms of action. This article is a PNAS Direct Submission. Moreover, mTORC1 controls tumor cell growth, and its activity Published under the PNAS license. is often dysregulated in cancer. This enzyme integrates diverse 1To whom correspondence may be addressed. Email: [email protected] or inputs, including amino acids, growth factors, and stress signals [email protected]. to regulate protein synthesis, autophagy, and nutrient metabolism This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (9–11). mTORC1 activity is controlled by 2 major small GTPases, 1073/pnas.1904774116/-/DCSupplemental. Rheb and Rag. Repression of mTORC1 by the depletion of specific First published September 23, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1904774116 PNAS | October 8, 2019 | vol. 116 | no. 41 | 20505–20510 Downloaded by guest on October 6, 2021 recognizes the motif “RXRXXS*/T*,” demonstrate that Pim1 is A B - Rag WT Rag CA Dox - Dox + capable of phosphorylating DEPDC5 (Fig. 2A). To validate these Leu - 0 2 4 6 02 4 6 h CPAAPCPPA AP C AAP phosphorylation sites, we generated a phospho-specific antibody P-S6K P-S6K T389 T389 against S1002 and purchased a commercially available S1530 an- 1 0.5 1.1 0.5 tibody. The specificity of the antibodies employed for these ex- S6K periments was validated using lysates of 293T cells expressing wild S6K P-S6 type (WT) and site-directed DEPDC5 mutants containing S1002A S240/244 P-S6 and S1530A (Fig. 2 B and C). Both mutant and WT DEPDC5 S6 S240/244 1 0.5 0.3 0 were transfected into 293T cells with and without Pim inhibitor. Pim1 S6 Pim inhibition reduced the phosphorylation of both S1002 and S1530 sites, suggesting that both sites are Pim targets (Fig. 2D). To RagB Tubulin examine AKT activity, we cotransfected the plasmids encoding Tripz Pim1 RagD a constitutively active form of AKT, myristoylated (myr) AKT P-IRS1 S1101 and DEPDC5 into 293T cells. Results demonstrate that AKT P-Foxo3a phosphorylates only the S1530 site on DEPDC5 and not S1002 S318/321 E P-GSK3ß (Fig. 2 ). S9 To examine whether endogenous Pim phosphorylates DEPDC5 GSK3ß in breast tumor cells, BT549 and MDA-MB231 TNBC cell lines Tubulin stably expressing Flag-DEPDC5 were developed. These specific TNBC cell lines were chosen as they have been shown to express Fig. 1. Pim kinases regulate amino acid mediated mTORC1 activation. (A) high levels of the Pim1 kinase (SI Appendix, Fig. S1A) (2, 3). Time course of leucine deprivation (Leu−) with and without Pim1 over- Importantly, the combination of a Pim and an AKT inhibitor expression performed in PC3-LN4 cell line containing Dox-inducible Pim1 (Tripz synergistically decreased the DEPDC5 phosphorylation at the Pim1) vector, treated with and without 100 ng/mL of Dox for 18 h. (B)PC3-LN4 S1530 site, whereas the S1002 site is specifically decreased by Pim control, overexpressing Rag WT or constitutively active Rag (CA) (see SI Ap- inhibitor treatment (Fig. 2F). Both inhibitors were capable of μ pendix, SI Materials and Methods) cells were cultured with DMSO (C), 3 M inhibiting mTORC1, as evidenced by a decrease in P-S6, as well as Pim447 (P), 5 μM GSK690693 (A), and the combination (AP) for 6 h. Cells were β lysed and analyzed by Western blotting (WB). See SI Appendix, SI Materials specific targets IRS1 for Pim and GSK3 for AKT. Since both and Methods for WB quantification. Numerical values shown under the blot AKT and Pim are known to be activated by mitogens (26, 27), we are calculated relative to the DMSO (C) treatment. examined whether a specific growth factor could activate DEPDC5 phosphorylation. The addition of insulin to serum-starved BT-549 cells stably expressing DEPDC5 activates both Pim and AKT ac- complex induces Rag dimers to switch to an inactive conformation tivity, as shown by IRS1 and GSK3β phosphorylation, and stimu- containing GDP-bound RagA/B, thereby inactivating mTORC1 lates a 1.8-fold increase in phosphorylation of DEPDC5 S1530 and (20).
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