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Home , ATG5, MTOR inhibitors, Proteasome inhibitor

mTOR inhibition activates overall degradation by the system as well as by

Jinghui Zhao, Bo Zhai, Steven P. Gygi, and Alfred Lewis Goldberg1

Department of Cell Biology, Harvard Medical School, Boston, MA 02115

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2015.

Contributed by Alfred Lewis Goldberg, November 6, 2015 (sent for review September 10, 2015; reviewed by David J. Glass and David Rubinsztein) Growth factors and nutrients enhance protein synthesis and sup- Rapamycin is a that selectively inhibits mTORC1 press overall protein degradation by activating the protein but not mTORC2. Torin1 is a synthetic mTOR inhibitor that blocks mammalian target of rapamycin (mTOR). Conversely, nutrient or ATP-binding to mTOR and thus inactivates both mTORC1 and serum deprivation inhibits mTOR and stimulates protein breakdown mTORC2 (7). Both Torin1 and rapamycin inhibit overall protein by inducing autophagy, which provides the starved cells with amino synthesis, induce autophagosome formation, and thus mimic the ef- for protein synthesis and energy production. However, it is fects of starvation. However, Torin1 is much more effective than unclear whether proteolysis by the ubiquitin proteasome system rapamycin in affecting these two processes because rapamycin in- (UPS), which catalyzes most protein degradation in mammalian hibits mTORC1 incompletely (7, 8). Because many types of cells, also increases when mTOR activity decreases. Here we show are associated with overactivation of mTOR, rapamycin and other that inhibiting mTOR with rapamycin or Torin1 rapidly increases the novel mTOR inhibitors are useful in the treatment of certain degradation of long-lived cell , but not short-lived ones, by (9). Rapamycin is widely used in the clinic as an immune suppressor stimulating proteolysis by , in addition to autophagy. (10).Nevertheless,rapamycinextendslifespaninagedmice,perhaps This enhanced proteasomal degradation required protein ubiquiti- by triggering similar changes as occur with dietary caloric restriction nation, and within 30 min after mTOR inhibition, the cellular content (11). Additionally, stimulating autophagy by rapamycin can help clear of K48-linked ubiquitinated proteins increased without any change intracellular protein aggregates as they accumulate in many neuro- in proteasome content or activity. This rapid increase in UPS-mediated degenerative diseases and reduce their (12). proteolysis continued for many hours and resulted primarily from Eukaryotic cells degrade proteins by both the autophagy-lysosome inhibition of mTORC1 (not mTORC2), but did not require new system and the ubiquitin proteasome system (UPS). Macroautophagy protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These delivers cytoplasmic proteins or organelles into autophagic vacuoles findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, for degradation, and autophagosome formation is rapidly activated et al. (2014) Nature 513(7518):440–443]. Several growth-related by starvation or mTOR inhibition (3). The UPS is responsible for the proteins were identified that were ubiquitinated and degraded degradation of most cytosolic and nuclear proteins in mammalian more rapidly after mTOR inhibition, including HMG-CoA synthase, cells, including the short-lived regulatory and misfolded proteins as whose enhanced degradation probably limits cholesterol biosyn- well as the bulk of cell constituents, which are long-lived components thesis upon insulin deficiency. Thus, mTOR inhibition coordinately (13, 14). Degradation by the UPS is highly selective, involving the activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic Significance proteins, help slow growth. Lack of nutrients or growth factors rapidly inhibits mTOR kinase mTOR | proteasome | ubiquitination | autophagy and stimulates overall protein breakdown by autophagy, which provides amino acids for new protein synthesis and energy pro- he balance between overall rates of protein synthesis and deg- duction. Here, we demonstrate that mTOR inhibition also rapidly Tradation determines whether a cell grows or atrophies. It has increases overall protein degradation by the ubiquitin proteasome long been proposed that overall rates of protein synthesis and deg- system by enhancing the ubiquitination of many cell proteins. radation can be coordinately regulated (1). For example, when These findings demonstrate a new general mechanism regulating hormones (e.g., insulin and insulin-like -1) and nutri- overall protein breakdown in mammalian cells. This rapid activation ents are plentiful, rates of protein synthesis are high and protein of proteolysis not only slows growth and provides essential amino degradation is suppressed, whereas in starving cells, synthesis falls acids upon starvation, but also stimulates ubiquitin-dependent and overall degradation rises. One critical factor coordinating overall degradation of certain growth-related proteins, including HMG- synthesis and degradation is the Ser/Thr mammalian CoA synthase, which is critical for cholesterol biosynthesis. target of rapamycin (mTOR), which promotes protein Accelerated degradation of such proteins represents a new (2) while suppressing autophagy (lysosomal proteolysis) (3). mechanism for rapid suppression of anabolic processes when mTOR’s pleiotropic functions are catalyzed by two distinct kinase mTORC1 activity decreases. complexes: mTORC1, the key component of which is Raptor, and Author contributions: J.Z. and A.L.G. designed research; J.Z., B.Z., and S.P.G. performed mTORC2, which instead contains Rictor (4, 5). Growth factors act research; J.Z. and A.L.G. analyzed data; and J.Z. and A.L.G. wrote the paper. through class I PI3K and Akt to inhibit the tumor suppressor Reviewers: D.J.G., Institutes for Biomedical Research; and D.R., University TSC2, and thereby activate mTORC1 (6). Adequate supply of of Cambridge. amino acids, especially leucine, can also activate mTORC1, but The authors declare no conflict of interest. through a distinct mechanism involving Rag GTPase and lysosomal 1To whom correspondence should be addressed. Email: Alfred_Goldberg@hms. recruitmentofmTORC1(4).UnlikemTORC1,mTORC2isin- harvard.edu. sensitive to amino supply but is activated by growth factors via This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mechanisms that remain unclear. 1073/pnas.1521919112/-/DCSupplemental.

15790–15797 | PNAS | December 29, 2015 | vol. 112 | no. 52 www.pnas.org/cgi/doi/10.1073/pnas.1521919112 Downloaded by guest on September 27, 2021 attachment of a ubiquitin (Ub) chain to the substrate through the conditions (18). In this study, we have used well-validated pulse-

sequential actions of a Ub-activating enzyme (E1), Ub-conjugating chase approaches (14) to measure proteolysis by proteasomal and INAUGURAL ARTICLE enzymes (E2s), and one of the cell’s many Ub ligases (E3s) (15). lysosomal systems after mTOR inhibition and have also monitored Proteins conjugated to Ub chains are rapidly degraded by the overall Ub conjugation and proteasome content. 26S proteasome, whereas the Ub molecules are recycled by protea- some-associated deubiquitinating enzymes (DUBs) (16). Although Results the degradation of specific short-lived proteins has been studied Inhibiting mTOR Rapidly Enhances the Degradation of Long-Lived but Not extensively, our understanding of the global mechanisms controlling Short-Lived Cell Proteins. WefirsttestedifmTORinhibitorsaffect proteasomal degradation of the bulk of cell constituents is the degradation of radiolabeled short-lived and more stable pro- very limited. teins in HEK293 cells by using pulse-chase approaches described The best-characterized function of mTOR is to enhance pro- previously (14). Treatment with either Torin1 or rapamycin en- tein translation through mTORC1-mediated of hanced the degradation of long-lived cell proteins (labeled for 20 h) 4E-BPs and S6Ks (2). The simultaneous suppression of protein reproducibly by 1 h, although Torin1 consistently caused a larger degradation is generally attributed to the ability of mTORC1 to increase (Fig. 1B). In contrast, neither agent increased the break- inhibit autophagy, which seems to occur by the phosphorylation down of short-lived proteins, which were degraded within 1 h after a and inactivation of Atg1/Ulks (3). The goal of this study was to brief labeling (20 min) (Fig. 1A). Therefore, subsequent studies test whether the increased proteolysis upon mTOR inhibition focused on the degradation of long-lived proteins, which comprise may also occur through activation of the UPS. We demonstrate the great majority of cell proteins. herein that mTOR inhibition not only enhances lysosomal pro- Torin1 inactivates both mTORC1 and mTORC2 (7), as was teolysis, but also rapidly stimulates the ubiquitination and confirmed by the elimination of phosphorylated S6K (Thr389) and proteasomal degradation of many proteins. We also investigated Akt (Ser473) (Figs. 2B,3F,and4C). However, rapamycin, which the mechanisms by which the UPS is activated, the role of inhibits only mTORC1, abolished S6K phosphorylation, while mTORC1 or mTORC2, and the nature of the proteins whose causing hyperphosphorylation of Akt (Fig. 2B and SI Appendix, stability is affected by mTOR. Fig. S6). Because rapamycin inhibits mTORC1 only partially (7), Together with the enhancement of autophagy, this activation of we mainly used Torin1 to clarify mTOR’s role, but also studied proteolysis by the UPS upon mTOR inhibition appears to represent rapamycin because of its wide use in research and the clinic. an important adaptation to starvation that helps slow growth and provide essential amino acids. A very different role of mTORC1 in Inhibiting mTOR Stimulates both Autophagy and Proteasome-Mediated BIOCHEMISTRY regulating protein degradation was recently proposed by Zhang Proteolysis. To determine whether mTOR inhibitors enhance protein et al. (17), who surprisingly reported that rapamycin did not rapidly breakdown by the UPS, we measured their effects on the degradation increase proteolysis, but after 16 h actually reduced overall pro- mediated by proteasomes or lysosomes by analyzing the fraction of teolysis by decreasing proteasome expression (17). Such a sup- overall proteolysis sensitive to the , pression is inconsistent with the well-established increase in (BTZ), or to the inhibitor of lysosomal acidification, concanamycin A proteolysis and autophagy in starving cells, and various observations (CCA), as described previously (14). As expected, Torin1 and to a about the regulation of the UPS (see below). Zhang et al.’s con- lesser extent rapamycin increased lysosomal (CCA-sensitive) pro- clusions probably result from their potentially misleading pulse- teolysis in both HEK293 cells and mouse embryonic fibroblasts chase methods to measure protein degradation and cell culture (MEFs) (Fig. 1D). The larger stimulation of lysosomal proteolysis by

Fig. 1. mTOR inhibitors enhance the degradation of ABSHORT-LIVED LONG-LIVED long-lived, but not short-lived, cell proteins by acti- 8 Torin1 vating proteasomal proteolysis, as well as autophagy. 20 (A) Rapamycin and Torin1 did not affect the degra- 6 Rapamycin dation of short-lived cell proteins in HEK293 cells. 15 Control Newly synthesized proteins were radiolabeled by in- 4 cubating cells in complete medium supplemented 10 with 3H-Phe for 20 min. After two quick washes of the cells with medium containing 2 mM nonradioactive 5 2 % Protein degraded Protein %

Phe and cycloheximide to prevent reuptake and rein- degraded Protein % corporation, control (♦,vehicle),rapamycin(□,300nM), 0 0 or Torin1 (▲, 100 nM) were added at time 0, and the 00.51 1.5 0241 3 5 6 Hours Hours release of 3H-Phe from cell proteins into the media was measured especially during the first hour CD when short-lived proteins are selectively degraded. Proteasomal degradation Lysosomal degradation (B) Rapamycin and Torin1 increased the degradation 1.0 * * Control of long-lived proteins in HEK293 cells. To label long- 1.2 * * 3 * Rap lived proteins selectively, cells were incubated with H- * 0.8 1.0 * * Torin1 Phe for 20 h. After switching to chase medium for 2 h * 0.8 to allow the degradation of short-lived components, 0.8 * 0.6 0.6 fresh chase medium containing mTOR inhibitors was 0.6 added, and proteolysis assayed. The fraction of ra- 0.4 0.4 * dioactive proteins degraded to TCA-soluble material 0.4 0.2 0.2 * at different times was expressed as a percentage of 0.2 total radioactivity incorporated into cell proteins at t = 0. Significant increases upon mTOR inhibition were 0 0 0 BTZ-sensitive proteolysis (%/h) proteolysis BTZ-sensitive HEK293 MEF MEF MEF (%/h) proteolysis CCA-sensitive HEK293 MEF MEF observed at 90 min and thereafter. (C) mTOR in- (%/h) proteolysis CCA-resistant (Atg5+/+) (Atg5−/−) (Atg5−/−) (Atg5+/+) (Atg5−/−) hibition with rapamycin or Torin1 increased degra- − − dation by proteasomes, which was measured as CCA-resistant proteolysis in HEK293 cells and MEFs (Left), or by BTZ-sensitive proteolysis in Atg5 / MEFs (Right). (D) mTOR inhibition increased lysosomal (CCA-sensitive) proteolysis in HEK293 cells and wild type MEFs, but not in Atg5−/− MEFs. Error bars (SEM) were provided in C and D and for every time point in A and B (but were hidden by labels in B). *P < 0.05.

Zhao et al. PNAS | December 29, 2015 | vol. 112 | no. 52 | 15791 Downloaded by guest on September 27, 2021 Torin1 is consistent with morphological evidence that Torin1 is more Activation of Proteasomal Degradation upon mTOR Inhibition Does effective than rapamycin in inducing autophagosome formation (7). Not Require Protein Synthesis. Although mTOR inhibition alters These increases in lysosomal degradation depend on autophagy, rates of translation (8) and transcription (20, 21), it rapidly stimulates because Torin1 failed to stimulate this process in MEFs unable to autophagy independently of protein synthesis (3). To learn whether form autophagosomes because of deficiency of Atg5 (Fig. 1D). the enhancement of proteasomal degradation by mTOR inhibitors In addition, Torin1 and rapamycin increased proteasomal requires protein synthesis, we pretreated HEK293 cells with cyclo- degradation, measured by either of two approaches: the CCA- heximide for 1 h before Torin1 addition. Although cycloheximide by resistant proteolysis in HEK293 cells and MEFs (Fig. 1C, Left) itself reduced proteasomal proteolysis [probably by enhancing or the BTZ-sensitive proteolysis in Atg5-null MEFs (Fig. 1C, mTOR activity (22)] after cycloheximide treatment, Torin1 caused a Right). In autophagy-competent cells, the CCA-resistant com- similar or greater increase in proteasomal proteolysis (Fig. 2A). ponent was used to measure proteasomal degradation (Fig. 1C, Thus, this rapid stimulation by mTOR inhibitors does not require Left) because we found that although BTZ does not directly new protein or mRNA synthesis. inhibit lysosomal function (19), it partially reduced the activation mTOR Inhibition Rapidly Stimulates K48-Linked Ub Conjugation to of autophagy by Torin1 (SI Appendix, Fig. S1) (probably by Proteins in Cells and in Vivo, Which Leads to Increased Proteolysis. blocking the degradation of an inhibitor of autophagy). Torin1 For most proteins, ubiquitination is the rate-limiting step for treatment similarly increased proteasomal proteolysis in both degradation by the UPS. We therefore tested if inhibiting mTOR undifferentiated and differentiated C2C12 cells (SI Appendix, enhances ubiquitination generally. Because newly synthesized Fig. S2). Therefore, mTOR inhibition in many cell types stimu- proteins account for a large fraction of the ubiquitinated proteins lates overall proteolysis independently of autophagy. in cells and mTORC1 influences their rates of synthesis, we In several cell types analyzed, proteasomes accounted for two- pretreated MEFs with cycloheximide for 1 h to focus on the thirds or more of the degradation of long-lived proteins, and ubiquitination of long-lived proteins. Although cycloheximide lysosomes for nearly all of the remainder (SI Appendix, Table decreased the total content of ubiquitinated proteins (Fig. 2C S1). Upon mTOR inhibition, although the relative increases in and SI Appendix, Fig. S11), addition of rapamycin or Torin1 for lysosomal proteolysis are much larger than that by the UPS 1 h resulted in a highly reproducible 30% increase in the total (especially with Torin1), proteasomes still accounted for a content of ubiquitinated proteins and a concomitant 30% re- greater or equal fraction of the total proteolysis than lysosomes duction in free Ub levels (Fig. 2B). This increase in Ub conju- (Fig. 1 C and D). gates was maximal after only a 30-min treatment and persisted

ABControl Rap: + ++− −−− − − 1.0 * Torin1 Torin1: −−− − −−+ ++ Fig. 2. mTOR inhibition rapidly increases the levels of 0.8 * 188 K48-linked Ub conjugates in cells and mouse liver without affecting proteasomal activity. (A)Thein- 0.6 Ub(P4D1): 98 crease in proteasomal degradation by Torin1 treat- 0.4 62 ment in HEK293 cells did not require protein synthesis. Proteasomal proteolysis was determined in the pres- 0.2 Ub(P4D1): 8 ence or absence of 20 μg/mL cycloheximide. (B)mTOR P-Akt(473): 0 inhibition increased the cellular content of Ub conju- Proteasomal proteolysis (%/h) Proteasomal proteolysis Vehicle CHX P-S6K(389): gates but not SUMO2/3 conjugates in MEFs, which β-actin: were pretreated with cycloheximide for 1 h and then with rapamycin or Torin1 for 1 h. Ub conjugates, free C CHX: −−−+ +++++ Ub, and SUMO2/3 conjugates were determined by Rap: + −−− ++− −− Control Western blot, and their intensities quantitated and Torin1: −−+ − −−−++ Rap β 140 ** Torin1 normalized to that of -actin. Phospho-S6K and -Akt were measured to confirm the efficacy of rapamycin 188 120 Ub and Torin1 treatments. (C) mTOR inhibition raised K48- 100 (K48): 98 linked, but not K63-linked, Ub conjugates in MEFs. 80 ** MEFs were pretreated with cycloheximide for 1 h and 60 then treated with rapamycin or Torin1 for 1 h. K48 or 188 40 K63-linked conjugates were determined by Western

Ub 20 blot with linkage-specific , and their in- 98 abundance Relative (K63): tensities quantitated and plotted in SI Appendix,Fig. 0 62 Ub Free Ub SUMO2/3 S11.(D) Rapamycin raised the level of Ub conjugates in Conjugates Conjugates mouse liver. CD1 mice (body weights ∼35 g) were in- jected intraperitoneally with 2.5 mg/kg rapamycin or β-actin: PBS. 2 h later, mice were killed, livers homogenized, and 100 μg total proteins were analyzed by Western blot. (E) Torin1 treatment did not increase peptidase Control DEControl activity of 26S proteasomes. HEK293 cells were first Mouse liver Rap 100 Torin1 treated with Torin1 or vehicle for 1 h before lysis, and Rap: − −−+ ++ 140 * 80 activity of 26S proteasomes in lysates was measured by 120 using the fluorogenic substrate Suc-LLVY-amc. Second, 188 60 100 we treated a mouse myoblast cell line that expresses a

Ub: 98 80 40 FLAG-tagged PSMB2 with Torin1 or vehicle for 1 h

(FL-76) of 26S activity before lysis. 26S proteasomes were then isolated with a 60 20 62 FLAG- resin, and peptidase activity of the pu- 40 Normalized peptidase 0 Lysate Purified rified 26S proteasomes measured. Western blots of 20 α-subunits of the proteasome are included to show

β-actin: of Levels Ub-conjugates α-subunits: 0 equal loading. *P < 0.05.

15792 | www.pnas.org/cgi/doi/10.1073/pnas.1521919112 Zhao et al. Downloaded by guest on September 27, 2021 ABProteasomal Lysosomal * Ube1 inhi: − + INAUGURAL ARTICLE 0.8 * Control * Torin1 0.6 188 Ub: 98 0.4 (FK2) 62 0.2 Fig. 3. The increased proteasomal degradation by Torin1 requires ubiquitination, but not Akts, S6Ks, and β-actin:

Rate of (%/h) proteolysis 0 Ulks, and results primarily from mTORC1 inhibition. Ube1 inhi: −−+ + (A) The increase in proteasomal degradation induced by mTOR inhibition requires ubiquitination. Proteasomal CD Eand lysosomal proteolysis were determined in the pres- Proteasomal Lysosomal Control ence or absence of 10 μM Ube1 inhibitor in MEFs. 0.12 * 0.10 * 0.5 Akti-1/2 * * Torin1 (B) Blocking ubiquitination markedly reduced the cellular 0.10 * 0.08 0.4 1.0 content of Ub conjugates. MEFs were treated with the 0.08 Ube1 inhibitor or vehicle for 1 h before lysis, and the 0.06 0.3 * 0.8 0.06 * levels of Ub conjugates was measured by Western blot 0.04 0.6 with anti-Ub (FK2). (C) The increase in proteasomal pro- 0.04 0.2 (%/h) 0.02 0.4 teolysis by Torin1 was independent of S6Ks or 4E-BPs. 0.02 0.1 Proteasomal proteolysis was determined in wild-type 0 0.2 Increase in proteasomal Increase MEFs and MEFs lacking 4E-BP1 and 2 or S6K1 and 2. 0 Rate of (%/h) proteolysis proteolysis by by Torin1 proteolysis (%/h) 0

Proteasomal proteolysis 0 (D) Overexpression of Ulk1 or Ulk2 enhanced lysosomal but not proteasomal degradation. Proteolysis was de- termined in HEK293 cells after 20 h of transfection with F I vectorsexpressingUlk1,Ulk2,oraninactivemutantof 6h + CHX: Akti-1/2 Vehicle Torin1 100 * n.s. n.s. Ulk1 (Ulk1-K46N). (E) Unlike Torin1, the Akt inhibitor, μ HMGCS1: 80 1.2 Akti-1/2, (2 M) did not increase proteasomal proteolysis in MEFs. (F) Akt inhibition did not induce the degrada- SUPT6H: 60 1.0 * tion of HMGCS1 and SUPT6H. The contents of these P-S6K(389): BIOCHEMISTRY 40 0.8 proteins were measured by Western blot after treat- P-Pras40: 0.6

20 (%/h) ments for 6 h in the presence of cycloheximide. Phospho- β-actin: * 0.4 Pras40 was measured to confirm the efficacy of Akti-1/2.

Relative protein levels Relative 0 HMGCS1 SUPT6H 0.2 (G) Torin1 increased proteasomal degradation in mTORC2-deficient (Rictor-null) MEFs. (H) Rictor-null GH proteolysis Proteasomal 0 Control Torin1: + − −−+ MEFs showed no mTORC2 activity, reduced mTORC1 Torin1 Rictor +/+ Rictor −/− Insulin: −−− + + activity, and similar levels of HMGCS1, SUPT6H, and * Medium:Complete EBSS α 1.2 * Rictor: -taxilin proteins compared with wild-type MEFs. (I) 1.0 Selective activation or inhibition of mTORC2 did not P-Akt(473): P-S6K(389):: affect proteasomal degradation in HEK293 cells. 0.8 P-S6K(389): P-Akt(473):: Proteasomal proteolysis was measured as described 0.6 HMGCS1: Akt:: in Methods. For EBSS treatment, cells were washed SUPT6H: 0.4 once with EBSS and incubated with EBSS plus or α-taxilin: 0.2 minus insulin for 30 min, and then Torin1 was added β-actin: < 0 for 1 h before measuring proteolysis. *P 0.05; n.s.,

Proteasomal proteolysis (%/h) Proteasomal proteolysis Rictor+/+ Rictor−/− not significant.

for at least 3 h (SI Appendix, Fig. S3B). Similar increases in Ub (Fig. 3A). Although blocking ubiquitination also reduced slightly the conjugate levels were seen in HEK293 cells using three different increase in lysosomal proteolysis by Torin1, about 70% of the Torin1- Ub antibodies (FL-76, FK2, P4D1) (SI Appendix, Fig. S3A) and induced activation of lysosomal degradation was independent of no change was observed in the levels of SUMO2/3 conjugates ubiquitination (Fig. 3A). Thus, the increased protein ubiquitination (Fig. 2B and SI Appendix, Fig. S3C). after mTOR inhibition is required for the accelerated proteasomal Ub chains are formed through isopeptide linkages between the degradation of long-lived proteins. We also tested for a possible C-terminal glycine of Ub and one of the lysines on the preceding additional effect of Torin1 in stimulating proteasomal activity, but Ub. Although K48-linked Ub chains target proteins to 26S pro- could detect no change in 26S proteasomal peptidase activity in teasomes, K63-linked chains do not (23). Using antibodies specific HEK293 extracts or after purification of proteasomes from the for these two linkage types, we found that rapamycin and Torin1 treated cells (Fig. 2E). In addition, we did not detect any effect of increased the cellular content of K48-linked Ub chains, but not of mTOR inhibitors on overall deubiquitinating activity in HEK293 K63-chains (Fig. 2C and SI Appendix,Fig.S11). To learn whether extracts, measured using the model substrate, Ub-amc (SI Appendix, mTOR inhibition also increases the level of Ub conjugates in vivo, Fig. S4A), nor did we observe any change in the activities of many we injected rapamycin into mice intraperitoneally and removed the individual DUBs measured by their modification with the active site livers 2 h later. An ∼30% similar increase in Ub conjugate levels probe, HA-Ub-vinyl sulfone (SI Appendix,Fig.S4B). was observed in liver extracts (Fig. 2D). This rapid increase in K48-linked Ub conjugates can account for mTOR Inhibition Increases Ubiquitination and Degradation of Growth- the enhanced degradation by proteasomes after mTOR inhibition Related Proteins. To identify some of the long-lived proteins whose and must reflect increased ubiquitination of many cell proteins. ubiquitination is stimulated by mTOR inhibition, we pretreated Accordingly, treating MEFs with a specific inhibitor of the major Ub- HEK293 cells with cycloheximide andthenwithTorin1orvehicle activating enzyme Ube1 (24) depleted the MEFs of nearly all Ub for 1 h. We then isolated ubiquitinated proteins from the lysates conjugates in 1 h (Fig. 3B) and reduced proteasomal proteolysis by at using immobilized tandem-repeated Ub-binding entities (25) and least 60% (Fig. 3A). Importantly, inhibition of Ube1 completely analyzed the conjugates by mass spectrometry (Fig. 4A). Many eliminated the enhancement of proteasomal degradation by Torin1 proteins were identified but showed little or no change in their

Zhao et al. PNAS | December 29, 2015 | vol. 112 | no. 52 | 15793 Downloaded by guest on September 27, 2021 ABLysis of HEK293 cells treated with vehicle or Torin1 for 1 hour Spectral counts by MS Protein names Exp-1 Exp-2 Enrichment of ubiquitinated proteins Control Torin1 Control Torin1 by immobilized GST-TUBE HMGCS1 29918 Release of ubiquitinated proteins from GST by PreScission protease cleavage SUPT6H 3 725 α-taxilin 8 13 3 11 Identification of ubiquitinated proteins by mass spectrometry Myst2 0 3 1 4

C Torin1: − − − +++ + + + −− BTZ: CHX 6h − −− −−− + −−− + 120 CCA: − −− −− − − + − − + CHX+Torin 6h MLN4924: −−−− −− −−+ −− 100 * HMGCS1: 80 # SUPT6H: 60 # α-taxilin: protein levels 40 Myst2: # 20 P-S6K(389): Relative 0 P-Akt(473):

β-actin: Fig. 4. Identification of proteins that are ubiquitinated and degraded more rapidly after Torin1 treatment. (A) Protocol for identifying proteins with increased D F ubiquitination after Torin1 treatment of HEK293 cells. BTZ Torin1 T+BTZ EBSS E+BTZ Insulin/IGF-1 (B) Examples of the 55 proteins with increased ubiquiti- 36 36363636(h) nation after Torin1 treatment, as shown by increased HMGCS1: number of peptides identified by mass spectrometry of 1.0 1.0 1.0 0.7 0.4 0.9 0.9 0.6 0.2 1.0 1.0 Amino PI3K ubiquitinated proteins. (C) Torin1 treatment increased Acids β-actin: the proteasomal degradation of 4 of the 12 proteins for which antibodies were obtained. Western blot of cell AKT extracts after treatments of MEFs for 6 h in the presence of cycloheximide, and their levels were quantified. E * Control (D) The marked reduction in HMGCS1 protein levels af- 0.8 * Torin1 mTOR ter treatment with Torin1 or EBSS buffer for 3–6hwas 0.6 prevented by BTZ treatment. (E) Torin1-caused increase in proteasomal proteolysis did not require a cullin-based 0.4 μ

(%/h) Ub ligase. MLN4924 (2 M) was used to inactivate all cullin-based Ub ligases. (F) Summary of the regulation of 0.2 Protein Autophagy Ubiquitination & proteasomal and autophagic proteolysis by mTOR, 0 synthesis Proteasomal downstream of nutrient and growth factor signaling. Proteasomal proteolysis Vehicle MLN4924 proteolysis *P < 0.05; #P < 0.01.

ubiquitination following mTOR inhibition, as determined by by three mechanisms: decreased transcription and translation, which counts of peptides recovered (SI Appendix,TableS2). However, we affect enzyme levels only slowly (because of its long half-life), and by identified 55 proteins for which at least two peptides were detected accelerated degradation, which allows enzyme levels to change much (see examples in Fig. 4B; SI Appendix,TableS3) that showed at faster. Accordingly, we observed a marked reduction in HMGCS1 least a 50% increase in ubiquitination after Torin1 treatment in two levels upon Torin1 treatment or deprivation of growth factors and independent experiments and we tested whether mTOR inhibition amino acids (EBSS buffer) for 3–6 h that could only occur through also enhances their degradation. Antibodies were available for 12 of enhanced degradation, not by reduced synthesis. In fact, BTZ the proteins that showed increased ubiquitination, and Torin1 completely prevented this fall in HMGCS1 content by Torin1 or treatment in the presence of cycloheximide reproducibly decreased EBSS buffer (Fig. 4D). This finding of accelerated degradation of the levels of four proteins, including HMGCS1 (cytoplasmic HMG- HMGCS1 after mTOR inhibition represents a new mechanism for CoA synthase), SUPT6H, α-taxilin, and Myst2 (Fig. 4C). Their rapid inhibition of the biosynthesis of cholesterol and isoprenoids accelerated degradation was mediated by proteasomes because upon nutrient or insulin deficiency. these decreases were blocked by BTZ, but not by CCA (Fig. 4C). The other three proteins also seem likely to have functions For the other eight proteins tested, mTOR inhibition did not sig- important for : transcription elongation and mRNA nificantly alter their degradation within 6 h (SI Appendix,Fig.S5). processing for SUPT6H, vesicular trafficking for α-taxilin, and Among these four proteins, the degradation of HMGCS1 was α stimulated to the largest extent by Torin1, which decreased its histone for Myst2. The contents of -taxilin and Myst2 – half-life from >10 h to about 2.5 h (SI Appendix, Fig. S6). In the are elevated in several tumors (29 32) and some proliferating mevalonate pathway for synthesis of cholesterol and isoprenoids, normal cells (33, 34). However, we could only examine the sta- HMGCS1 precedes HMG-CoA reductase (26). Inhibition of bilities of 12 proteins, and many ubiquitinated proteins must have mTORC1 reduces the transcription of HMGCS1 and other en- been missed by this approach because of their low abundance, zymes in this pathway through SREBP2 (sterol regulatory element- rapid degradation, or incomplete isolation. Most likely, these four binding protein 2) (20, 27, 28), and we found that Torin1 also represent only a small fraction of the proteins whose ubiquitina- rapidly decreased the synthesis of HMGCS1 by 25% (SI Appendix, tion and degradation are suppressed by mTOR. Stabilizing key Fig. S7). Therefore, mTOR inhibition reduces levels of HMGCS1 anabolic proteins along with the reduced ubiquitination of cell

15794 | www.pnas.org/cgi/doi/10.1073/pnas.1521919112 Zhao et al. Downloaded by guest on September 27, 2021 proteins generally seem to represent additional new mechanisms observed until 16 h of rapamycin treatment, in sharp contrast to the

by which mTOR promotes growth. rapid increase in proteasomal degradation described here and the INAUGURAL ARTICLE rapid activation of autophagy observed by many prior investigators The Enhancement in Proteasomal Proteolysis Does Not Involve mTOR (3). Their proposed mechanisms are also inconsistent with a num- Targets 4E-BPs, S6Ks, Ulks, or Akt. Because the increases in ber of prior findings about the UPS. Proteasomes are long-lived cell proteasomal proteolysis and autophagy upon mTOR inhibition constituents (subunit half-lives of 40–200 h) (35). Thus, simply de- occur simultaneously with the decrease in protein synthesis, we creasing proteasome transcription should not reduce their investigated whether the mTOR targets that activate translation or content within a short period. In fact, after rapamycin treatment for inhibit autophagy might also regulate proteolysis by the UPS. S6Ks 24 h, we did not detect any significant decrease in the levels of and 4E-BPs mediate the stimulation of translation by mTORC1 (2). several proteasome subunits (SI Appendix,Fig.S9)orin26Sactivity However, neither appears to be important for effects on proteasomal (peptidase and Ub conjugate degradation) (Fig. 2E). Furthermore, proteolysis, because Torin1 treatment of MEFs lacking S6K1 and proteasomes appear to be present in excess in cells (36), and their S6K2 or lacking 4E-BP1 and 4E-BP2 increased proteasomal pro- amount is usually not rate-limiting for proteolysis. In fact, we teolysis as in wild-type MEFs (Fig. 3C). Ulk1 and Ulk2 are critical demonstrated recently that a modest (∼30%) reduction of 26S for the suppression of autophagy by mTORC1 (3). Although over- proteasome content by knocking down PSMD2 has no effect on expression of either Ulk1 or Ulk2, but not the catalytically inactive overall protein degradation by proteasomes (37). Most importantly, mutant Ulk1-K46N, stimulated proteolysis by lysosomes, these using the same cells they studied, we did not find any decrease in treatments did not affect proteasomal degradation (Fig. 3D). Be- proteolysis, but only that mTOR inhibition activates overall pro- cause Akts are substrates of mTORC2 and activate many growth- teolysis rapidly and for at least 28 h (18). related processes, we tested if Akts are also important in suppressing As discussed elsewhere (18), both the pulse-chase analysis and proteasomal proteolysis. However, inhibition of Akts with Akti-1/2, experimental design of Zhang et al. (17) appear potentially as confirmed by the loss of phosphorylated Pras40 (Thr246) (Fig. misleading. In our experiments, we always maintained cells in 3F), did not increase proteasomal proteolysis generally (Fig. 3E), nor complete medium and compared the effects of mTOR inhibition the degradation of HMGCS1 and SUPT6H (Fig. 3F). Although the on degradation of the exact same pool of radiolabeled proteins. simultaneous stimulation by mTOR of translation and the inhibition However, their experiments compared the breakdown of proteins of autophagy and proteasomal proteolysis are coordinated responses labeled in TSC2-null, TSC2-expressing, or rapamycin-treated MEFs, that together promote protein accumulation, they are signaled by where patterns of transcription and translation differ as a result of

distinct mechanisms. differences in mTORC1 activity (17), and thus, they have compared BIOCHEMISTRY the degradation of distinct sets of proteins and not just study The Increase in Proteasomal Degradation Results Mainly from mTORC1’s effects on the proteolytic machinery. Additionally, Inhibition of mTORC1. To determine whether the enhancement Zhang et al.’s experiments involved depriving TSC2-null MEFs of of proteasomal proteolysis by Torin1 is caused by an inhibition of serum for extended periods (up to ∼66 h) (17) to study mTORC1’s mTORC1 or mTORC2, we performed two types of experiments. actions separately from mTORC2’s. However, this approach is First, in Rictor-null MEFs that lack mTORC2 activity (Fig. 3H), problematic for studies of proteolysis because serum deprivation by Torin1 treatment still increased overall proteasomal degradation itself stimulates proteolysis in all cells, and based on the present (Fig. 3G) and enhanced HMGCS1 degradation as in wild-type findings should do so more in wild-type cells than in TSC2-null MEFs (SI Appendix, Fig. S8). Thus, mTORC1 alone stabilizes MEFs. In fact, when we measured the breakdown of the same set of many cell proteins. Second, when HEK293 cells were deprived proteins in TSC2-null MEFs cells after serum deprivation for 17 h, of both serum and amino acids, which inactivated completely the subsequent inhibition of mTORC1 by rapamycin or Torin1 still mTORC1 activity and reduced significantly mTORC2 activity, increased overall proteolysis (SI Appendix,Fig.S10), as we consis- proteasomal proteolysis increased (Fig. 3I). However, addition of tently found in complete medium. Furthermore, long-term mTOR insulin, which activated strongly mTORC2 but not mTORC1, did inhibition or serum deprivation may trigger secondary responses. In not reduce the enhancement of proteasomal proteolysis, and fact, although serum deprivation acutely activates autophagy, long- inhibiting this hyperactivation of mTORC2 by Torin1 did not en- term serum deprivation or sustained inactivation of insulin-like hance it either (Fig. 3I). Taken together, the ability of Torin1 to growth factor 1 signaling reduces autophagy (38). Therefore, we activate proteasomal proteolysis in the absence of mTORC2 (Fig. focused in this study on the effects of mTOR inhibition for minutes 3G) and the lack of effect of mTORC2 activation or inhibition or several hours. alone (Fig. 3I) indicate that in HEK293 cells and MEFs, mTORC1 plays the major role in suppressing overall degradation by the UPS. The Potential Mechanisms by Which mTORC1 Suppresses Proteasomal Degradation. Our various experiments indicate that the activation by Discussion mTOR inhibition of protein breakdown by the UPS results primarily This New General Mechanism Controlling Protein Turnover and the from inhibition of mTORC1 and is driven by increased ubiquitination Conflicting Report. The present studies demonstrate that in nutrient- without any demonstrable activation of the proteasomes. In several rich environments and during growth, mTOR (primarily mTORC1) prior reports, mTOR-mediated phosphorylation of proteins was decreases overall protein degradation by the UPS and stabilizes many shown to influence their ubiquitination and stability. For example, normally long-lived proteins by suppressing their ubiquitination. Grb10, a negative regulator of growth factor signaling, was reported Under these conditions, mTOR does not seem to affect the break- to be stabilized by mTORC1 (39, 40). Among the four proteins we down of short-lived proteins (i.e., misfolded proteins or highly regu- identified that show accelerated degradation upon mTOR inhibition, lated proteins, whose ubiquitination has been studied most SUPT6H was reported to be a potential substrate of mTOR. Among extensively). When mTOR levels fall, upon lack of nutrients or the 55 proteins we identified that show increased ubiquitination after insulin, this mechanism can rapidly enhance (within 30–60 min) mTOR inhibition, 9 were reported to be tentative mTOR substrates the supply of amino acids without requiring alterations in tran- (39, 40). Therefore, mTORC1-mediated phosphorylation of certain scription or translation. proteins may lead to their stabilization by inhibiting their ubiquiti- As we were preparing this report, opposite findings were reported nation, and this mechanism may contribute to the general increases by Zhang et al. (17), who concluded that rapamycin treatment for in Ub-dependent proteolysis upon mTOR inhibition. Another pos- 16 h or longer reduces overall proteolysis by decreasing proteasome sibility is that mTOR directly inhibits certain ubiquitinating enzymes expression through decreases in the expression of the transcription or stimulates DUBs, and thus influences the degradation of some factor, Nrf1 (17). Surprisingly, no change in rates of proteolysis was proteins. However, we have not succeeded in finding such enzyme

Zhao et al. PNAS | December 29, 2015 | vol. 112 | no. 52 | 15795 Downloaded by guest on September 27, 2021 candidates. Possibly, multiple mechanisms downstream of mTORC1 mTORC1 are simultaneous and linked, they involve distinct down- (e.g., phosphorylation of substrates, Ub ligases, or DUBs) together stream mechanisms, because mTORC1’s suppression of the UPS is account for the increases in ubiquitination and proteasomal pro- independent of its targets that control translation or autophagy. This teolysis upon mTOR inhibition. It will clearly be important to learn rapid activation of the UPS may also synergize with the activation of which Ub ligases are responsible for the increased ubiquitination of autophagy in helping eliminate abnormal toxic proteins and thus the mTOR-regulated proteins, such as HMGCS1. A large fraction of contribute to the beneficial effects of rapamycin in models of neu- the Ub ligases in the belong to cullin-based protein rodegenerative disease (12) and in extending lifespan (11). complexes, many of which modify phosphorylated proteins se- lectively. However, these cullin-based Ub ligases appear not to Methods be involved, because exposing cells to the selective neddylation Cell Culture and Materials. HEK293, MEFs, and C2C12 myoblasts were main- inhibitor MLN4924 (41), which inhibits their activities, did not tained in DMEM with 10% (vol/vol) FBS in 5% (vol/vol) CO2 at 37 °C. C2C12 inhibits the degradation of these four proteins (Fig. 4C), nor myotubes were obtained by incubating C2C12 myoblasts with differentia- the increase in overall proteasomal proteolysis by Torin1 (Fig. tion medium [DMEM with 2% (vol/vol) horse serum] for 6 d in 8% (vol/vol) 4E). Thus, the responsible Ub ligases belong to the monomeric CO2 at 37 °C. Rapamycin and Akti1/2 were purchased from Calbiochem, con- canamycin A from Santa Cruz, and HA-Ub-vinyl sulfone from Boston Biochem. RING or HECT family. All mouse experiments were performed with the approval of the Harvard Although cells contain a family of proteins that bind ubiquiti- Medical School Institutional Animal Care and Use Committee and are in nated proteins and can target them to autophagic vacuoles for accordance with the Guide for the Care and Use of Laboratory Animals (48). degradation (42), it seems most likely that a great majority of proteins showing increased ubiquitination upon mTOR inhibition Determination of Total, Proteasomal, and Lysosomal Protein Degradation. This aredegradedbythe26Sproteasome.AsshowninFig.3A,the approach was described in detail and validated previously (14). Briefly, cells Torin1-induced increase in lysosomal proteolysis was largely un- were incubated with 3H-Phe (5 μCi/mL) in standard culture medium for 20 h affected by blocking Ub conjugation, whereas the increase in to label long-lived cell proteins, and then switched to a chase medium for 2 h proteasomal proteolysis required ubiquitination. In addition, the that contains 2 mM nonradioactive Phe to prevent reincorporation of re- leased radioactive amino acids. The medium was replaced with fresh chase four proteins identified as undergoing accelerated ubiquitination μ upon Torin1 treatment were all degraded by proteasomes and medium containing the vehicle, BTZ (1 M), or CCA (100-200 nM). One hour later, the mTOR inhibitors were added for another 1 h. Then multiple not lysosomes (Fig. 4C). Thus, the stimulation of autophagy and samples of the medium were collected at different times (up to 2 h) and proteasomal degradation differs in their dependence upon mixed with trichloroacetic acid [TCA, final 10% (vol/vol)] to precipitate ubiquitination, and the increase in protein ubiquitination probably proteins. The TCA-soluble radioactivity in the medium at different times drives the enhanced proteasomal proteolysis. reflects the amount of prelabeled, long-lived proteins degraded and was Although the degradation of many individual proteins by the expressed relative to the total radioactivity initially incorporated into pro- UPS has been extensively studied, global regulation of pro- tein. We generally used CCA-sensitive proteolysis to evaluate lysosomal teolysis by this pathway has received little attention, even though degradation and CCA-resistant proteolysis to evaluate proteasome-medi- a general stimulation of degradation by the UPS is critical in ated protein breakdown. All measurements were performed at least in “ ± ” catabolic states, such as muscle atrophy (43). Recently, a very triplicate, and the calculated rates of proteolysis are shown as mean SEM. P values were determined by two-tailed Student’s t test. different type of global regulation of the UPS has been described in which proteasome function is activated by cAMP and PKA- Western Blotting. Cell proteins were extracted in lysis buffer [1% Triton X-100, dependent phosphorylation, where proteolysis rises without any 10 mM Tris pH 7.6, 150 mM NaCl, 30 mM Na pyrophosphate, 50 mM NaF, 5 mM

increase in total ubiquitination (44, 45). It is noteworthy that EDTA, 0.1 mM Na3VO4 and protease inhibitor mixture (Roche)]. When mea- these two very different biochemical mechanisms also have distinct suring the levels of Ub conjugates, N-ethylmaleimide (10 mM) was added to the physiological roles and substrates. The PKA-mediated phosphory- lysis buffer to inactivate most DUBs. Thirty micrograms of total proteins were lation of proteasomes stimulates selectively the degradation of separated by SDS/PAGE, transferred to PVDF membranes, and analyzed by short-lived regulatory or misfolded proteins (e.g., aggregation-prone Western blot using the ECL method (Amersham). We used antibodies against proteins causing neurodegenerative diseases) (44, 45), whereas Akt, P-S6K (Thr389), P-Akt (ser473), P-Pras40 (T246), β-actin, α-tubulin, and Rpn11 from ; HMGCS1, P4D1, FL-76, and α-taxilin from Santa Cruz; mTOR affects the breakdown by the UPS of long-lived compo- α nents, which comprise the bulk of cell proteins and thus must be Rictor and SUPT6H from Bethyl; FK2, Rpt5, -subunits from Biomol. Western blot results were quantified by using ImageJ. important in growth regulation and adaptation to starvation. Isolation and Mass Spectrometry Analysis of Ubiquitinated Proteins. Twelve Coordinate Regulation of the UPS and Autophagy in Growing and 150-mm dishes of HEK293 cells were pretreated with cycloheximide for 1 h Starving Cells. Traditionally, the UPS and the lysosomal pathway and then with either vehicle or Torin1 for another 1 h. After cell lysis in the havebeenassumedtofunctionindependently, and autophagy acti- presence of 10 mM N-ethylmaleimide to block deubiquitination, 800 μgof vationhasbeenviewedasacompensatorymechanismwhenpro- GST-UBA proteins were added to the lysates and incubated for 4 h and then teasome function is inhibited (46). However, our prior studies with glutathione-resin for another 2 h. The immobilized proteins were ex- demonstrated that in fasting, after denervation and in other catabolic tensively washed with PBS, digested with PreScission proteases overnight to states, the loss of muscle protein (atrophy) involves coordinate acti- release the ubiquitinated proteins together with the UBA domains from the vation of these two degradative systems through the activation of GST-resin. These soluble proteins were then separated by SDS/PAGE, diges- FoxO transcription factors (14, 47). Unlike mTOR inhibition, which ted with trypsin, and analyzed by nanoscale-microcapillary reversed-phase liquid chromatography tandem mass spectrometry (LC-MS/MS) (49). enhances proteolysis via the UPS and autophagy within minutes, the FoxO-mediated stimulation requires hours for transcription and ACKNOWLEDGMENTS. We thank Y. K. Ohsumi (Tokyo Institute Technology) translation, but can lead to prolonged activation of these two deg- for the generous gift of Atg5-deficient mouse embryonic fibroblasts; radative systems (14, 47). Most likely, upon starvation in vivo these N. Sonenberg (McGill University) for the generous gift of 4E-BP1/2-deficient complementary mechanisms for activating proteolysis, by mTOR mouse embryonic fibroblasts; G. Thomas (University Cincinnati) for the gen- erous gift of S6K1/2-deficient mouse embryonic fibroblasts; D. M. Sabatini inhibition and FoxO activation, function sequentially to enable cells (Massachusetts Institute of Technology) for the generous gift of Rictor- to mobilize essential amino acids for the synthesis of proteins nec- deficient mouse embryonic fibroblasts; N. S. Gray (Harvard) and D. M. essary for cell survival or energy production. Conversely, when nu- Sabatini (Massachusetts Institute of Technology) for the generous gift of trients and growth factors are ample, mTOR is activated, and FoxOs Torin1; S. Thomas (Harvard) for plasmids for Ulks; and Takeda Pharmaceuti- cals for bortezomib and the Ube1 inhibitor. This work was supported by inactivated, the coordinate reduction in both proteolytic systems National Institute of Health Grants 5R01AR055255 and 5R01GM051923 (to synergizes with the enhancement of protein synthesis to promote A.L.G.); and grants from the Muscular Dystrophy Association of America, proteins accumulation. Although these three anabolic actions of Packard Center, and ALS Association (all to A.L.G.).

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