Regulation of mTOR function
`in response to hypoxia by REDD1
`and the TSC1/TSC2 tumor
`suppressor complex
`
`Iarnes Brugarolas} Kui Lei,‘°‘ Rebecca L. Hurley,3 Brendan D. Manning,‘ Ian H. Reiling,5
`Ernst Hafen,5 Lee A. Witters,3 Leif W. Ellisen,2 and William G. Kaelin ]r.1'6'7
`‘Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115,
`USA; 2Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts 02116, USA,-
`3Departments of Medicine and Biochemistry, Dartmouth Medical School and Department of Biological Sciences, Dartmouth
`College, Hanover, New Hampshire 03755, USA; “Department of Genetics & Complex Diseases, Harvard School of Public
`Health, Boston, Massachusetts 02115, USA; 5Zoologisches Institut, Uriiversitaet Zuerich, Winterthurerstr. 190, CH-8057
`Zuerich, Switzerland; ‘Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA
`
`Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is
`modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy
`depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMI’Kl
`by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition byhypoxia is not
`known. Here we show that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex
`and the hypoxia-inducible gene REDD1/RTP801. Disruption of the TSC1/TSC2 complex through loss of TSC1
`or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets SGK and
`4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIP). In contrast to energy
`depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity
`by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the
`hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and
`REDD1 overexpression is sufficient to down-regulate SGK phosphorylation in a TSC1/TSC2-dependent
`manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as
`TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia.
`
`[K85/'Words: Tuberous Sclerosis Complex; TSC1; TSC2; REDD1/RTP80l; mTOR; Hypoxia]
`Supplemental material is available at http://www.genesdev.org.
`Received August 31, 2004; revised version accepted October 6, 2004.
`
`Tuberous sclerosis complex, a disease characterized by
`benign tumors in multiple tissues, results from muta-
`tions in either Tuberous Sclerosis Complex 1 (TSC1) or 2
`(Tsc2) (Cheadle et al. 2000). Tscl (also called hamartin]
`and TscZ [also called tuberin) form a protein complex
`(van Slegtenhorst et al. 1998} that integrates signals from
`a variety of sources, including growth factors [Gao and
`Pan 2001; Potter et al. 2001; Tapon et al. 2001) and en-
`ergy stores (Inoki et al. 2003b}, with the protein transla-
`tion apparatus. Tscl functions as a GTPase-activating
`protein (GAP) toward the small G protein Rheb, which
`through a poorly understood mechanism controls mam-
`
`7Corresponding author.
`E-MAIL william_kaelin@dfci.l1arvard.edu; FAX (617) 632-4760.
`Article published onlinc ahead of print. Article and publication date are
`at http://www.genesdev.org/cgi/doi/10.I101/gad.125-6804.
`
`malian target of rapamycin (mTOR), a central regulator
`of protein translation (Castro et al. 2003; Gararni et al.
`2003; Inoki et al. 200321; Saucedo et al. 2003; Stocker et
`al. 2003; Tee et al. 2003; Y. Zhang et al. 2003).
`Regulation of mTOR by growth factors has been in-
`tensively studied. In response to growth factor stimula-
`tion, phosphatidylinositol 3-kinase (PI3K) is activated,
`leading to the generation of phosphaticlylinositol-3,4,5-
`triphosphate and the recruitment of Akt to the plasma
`membrane where it
`is activated by phosphorylation
`(Cantley 2002). Akt is a serine/threonine kinase that
`phosphorylates many effectors including Tsc2 (Dan et al.
`2002; Inoki et al. 2002; Manning et al. 2002; Potter et al.
`2002.). The mechanism whereby Akt phosphorylation
`regulates Tsc2 function is controversial, but it is thought
`ultimately to lead to its inactivation, thereby allowing
`the accumulation of Rheb-GTP and the activation of
`
`GENES 8:. DEVELOPMENT 182893-2904 © 2004 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/04; www.genesdev.org
`
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`mTOR. In support of the idea that Tscl/Tsc2 plays a
`critical role in mTOR regulation by growth factors, cells
`deficient for Tscl or Tsc2 fail to down-regulate 1nTOR
`function in response to growth factor deprivation (Gao et
`al. 2002; Inoki et al. 2002; Iaeschlce et al. 2002; Kwiat-
`kowski et al, 2002; H. Zhang et al. 2003].
`Recent studies have also shed light on how Tscl/Tsc2
`regulates mTOR function in response to changes in en-
`ergy availability. AMP-activated protein kinase {AMPK}
`is a master regulator of energy metabolisrn that is acti-
`vated in response to energy deprivation (Carling 2004).
`AMPK functions as a serine/threonine kinase and di-
`rectly phosphorylates Tsc2 (Inoki et al. 2003b). Cells de-
`ficient for Tscl/Tsc2, or producing a TSC2 variant that
`can not be phosphorylated by AMPK, fail to down-regu-
`late mTOR in situations of energy deprivation (Inoki et
`al. 2003b). Signaling by energy depletion also involves
`the Lkbl tumor suppressor protein, which is inactivated
`in Peutz-Ieghers syndrome. Lkbl is a serine/threonine
`kinase that pbosphorylates a variety of substrates, in-
`cluding AMPK (Hawley et al. 2003; Woods et al. 2003;
`Shaw et al. 2004b). AMPK activation with consequent
`Tscl /Tsc2-mediated inhibition of mTOR by energy
`depletion requires Lkbl [Corradetti et al. 2004; Shaw et
`al. 2004b). Thus under conditions that are adverse for
`growth, such as in the presence of reduced energy stores,
`mTOR function is inhibited, thereby down-regulating
`protein synthesis and conserving energy.
`mTOR function is also regulated by amino acid avail-
`ability (Gingras et al. 2001}. Whereas mTOR regulation
`by growth factors and energy stores requires an intact
`Tscl /Tsc2 complex, amino acids seem to regulate
`mTOR function through both Tscl/Tsc2-dependent and
`independent pathways (Gao et al. 2002, H. Zhang et al.
`2003}.
`mTOR is a conserved serine/threonine kinase that
`phosphorylates a series of substrates involved in protein
`translation including 4E-BP1 and S6K (Gingras et al.
`2001). 4E—BPl binds the translation initiation factor eIF-
`4E, preventing its interaction with other members of the
`eIF-4 complex and thereby inhibiting translation initia-
`tion of 5’ cap (7-methyl GTP) mRNAs. 4E—BPl phos-
`phorylation by mTOR, as well as other kinases in the
`PI3K pathway, relieves this inhibition, thereby promot-
`ing mRNA translation (Gingras et al. 2001).
`In mammals, there are two 36K genes, 36K] and S6K2,
`and each has two different splice forms. While S6K2 and
`the long form (~85 l<Da) of S6Kl localize to the nucleus,
`the short form of S6Kl [~70 l<Da) is cytoplasmic. 86K is
`thought to exist in an inactive, closed conformation, re-
`sulting from an intramolecular interaction between the
`catalytic domain and a pseudosubstrate domain (Fingar
`and Blenis 2004). There are at least eight phosphoryla-
`tion sites in S6Kl and its activation requires a complex
`process of phosphorylation involving mTOR as well as
`other PIBK effectors. Phosphorylation in the linker re-
`gion, at Ser 371 and Thr 389, is essential for S6Kl acti-
`vation [Dufner and Thomas 1999). Thr 389 is the major
`rapamycin-sensitive site and can be phosphorylated by
`mTOR in vitro, suggesting that it is a direct mTOR tar-
`
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`GENES EL DEVELOPMENT
`
`get (Dufner and Thomas 1999). Phosphorylation at this
`site has also been shown to be regulated in response to
`changes in Tscl/Tsc2 levels [Inoki et al. 2002, Iaeschke
`et al. 2002; Kwiatkowski et al. 2002, Manning et al.
`2002; H. Zhang et al. 2003).
`The best-characterized substrate of S6Kl is the ribo-
`sornal protein S6. S6 is an integral component of the 40S
`subunit that is required for cell proliferation but whose
`precise function is unclear (Volarevic et al. 2000). S6 is
`thought to be regulated primarily through successive
`phosphorylation events beginning at S236 and S235,
`Phosphorylation at these sites is rapamycin-sensitive
`and largely mediated by SGK [Dufner and Thomas 1999).
`Indeed, cells deficient for both S6Kl and S6K2 have pro-
`foundly reduccd levels of S6 S235/236 phosphorylation
`(Pende et al. 2004). Thus, S235/236 phosphorylation pro-
`vides an accurate readout for endogenous 56K activity.
`The I-Iypoxiadnducible factor
`[Hif) has also been
`shown to be regulated by mTOR (Hudson et al. 2002).
`Hif is a heterodimeric transcription factor composed of a
`stable [3 and a labile or subunit whose levels are con-
`trolled by oxygen tension {Semenza 2000). In the pres-
`ence of oxygen, Hif-or subunits are hydroxylated at spe-
`cific prolyl residues and targeted for degradation by an E3
`ubiquitin ligase that contains the von Hippel-Lindau tu-
`mor suppressor protein (pVHL) (Kaelin 2002). pVHL in-
`activation in patients with von Hippel-Lindau disease
`results in Hif up-regulation and the development of tu-
`mors. As in pVHL-deficient cells, Tsc2-deficient cells
`harbor increased levels of Hif-or relative to wild~type
`cells, especially under growth factor poor conditions
`[Brugarolas et al. 2003). Hif up—regulation in Tsc2-defi-
`cient cells is likely to result from increased mTOR ac-
`tivity as mTOR increases Hif stability and increased Hif
`levels in Tsc2-deficient cells can be normalized by treat-
`ment with rapamycin [Hudson et al. 2002, Brugarolas et
`al. 2003}.
`It was recently shown that mTOR function is regu-
`lated by hypoxia (Arsham et al. 2003). Hypoxia down-
`regulates 4E—BPl phosphorylation and increases 4E-BP1
`binding to eIF-4E at 5’ cap structures. Similarly, hypoxia
`down-regulates S6K phosphorylation at multiple sites
`including T389 and inhibits S6 phosphorylation. Hy-
`poxia-induced inhibition of mTOR is dominant over
`mTOR activating signals from growth factors and nutri-
`ents and occurs independently of Hif—loz [Arsham et al.
`2003). However, how mTOR function is regulated by
`hypoxia is not known.
`Here we show that mTOR inhibition by hypoxia re-
`quires an intact Tscl/Tsc2 complex. Furthermore,
`down-regulation of mTOR function by hypoxia requires
`de novo transcription and the expression of the hypoxia-
`inducible Redd] /RTP801 gene.
`
`Results
`
`Tscl/Tsc2 complex is required for mTOR regulatiozi
`by hypoxia
`
`To examine the contribution of the Tscl/Tsc2 complex
`to the regulation of mTOR function by hypoxia, the ef-
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`fects of hypoxia on MEFs derived from Tsc2—deficient
`embryos were analyzed. Loss of Tsc2 results in lethality
`at approximately day 10.5 of gestation when it is diffi-
`cult to obtain a large number of fibroblasts (Rennebeck
`et al. 1998). To extend the life span of the few fibroblasts
`that can be obtained from these embryos, mice were in-
`tercrossed with mice carrying mutations in p53, which
`enhances the life span of the limited number of MEFs
`that can be obtained (H. Zhang et al. 2005). In the ex-
`periments that follow, Tsc2‘/‘,«p53'/‘ MEFS were com-
`pared with Tsc2*/‘;p53“/‘ MEFs. For simplicity, these
`MEFs are referred to as ”Tsc2‘(‘” and ”Tsc2*/*,” respec-
`tively.
`Tscl/Tsc2 complex regulates mTOR in response to
`growth factors. As shown before, serum deprivation in-
`hibits mTOR, as evidenced by decreased phosphoryla-
`tion of the mTOR effector S6K (T389) (Fig, 1A). S6K
`T389 is the major rapamycin-sensitive site and is re-
`quired for S6K activity (Dufner and Thomas 1999). The
`down-regulation of 56K phosphorylation is associated
`with a decrease in S6 phosphorylation (S235/236). We
`also confirmed that mTOR inhibition by serum depri-
`vation requires an intact Tscl/Tsc2 complex and is
`abrogated in T302‘/' MEFS (Fig. 1A) (Iaeschke et al. 2002;
`H. Zhang et al. 2003). Rapamycin inhibits S6K phos-
`phorylation and activity regardless of Tse2 status, sup-
`porting the concept that Tscl/Tscl functions upstream
`of mTOR (Fig. IA),
`Tsc2*/’ MEFS down-regulated S6K phosphorylation
`and activity in response to hypoxia (Fig. IA), in keeping
`
`Hypoxia regulation of mTOR by REDDI and TSC1/TSC2
`
`with an earlier study using transformed human embry-
`onic kidney [HEK293) cells (Arsharn et al. 2003). Down-
`regulation of S6 phosphorylation in response to hy-
`poxia was also demonstrable in mice exposed to 6% oxy-
`gen, suggesting that this phenomenon is not restricted to
`tissue culture (Supplementary Fig. 1). In contrast, hy-
`poxia failed to down-regulate S6K and S6 phosphoryla-
`tion in Tsc2‘(‘ MEFs (Fig. IA) and Tscl‘/‘ mouse 3T3
`cells (Fig. 113), indicating that mTOR inhibition by hy-
`poxia requires an intact Tscl/Tsc2 complex. Likewise
`hypoxia promoted the binding of 4E—BP1 to 7—methyl
`GTP (7rnGTP) in T.sc2*/* cells but not Tsc2‘/” cells, con-
`sistent with Tsc2—dependent inhibition of 4E-BP1 phos-
`phorylation by mTOR in response to hypoxia (Fig. 1C).
`Taken together, these data indicate that mTOR inhibi-
`tion by hypoxia requires a functional Tscl/Tsc2 com-
`plex.
`We previously showed that Hif-oi (hereafter referred to
`as Hif) levels are regulated in response to growth factors
`through a mechanism that involves the Tscl/Tsc2 com-
`plex and inTOR (Brugarolas et al. 2003; see also Fig. 1A).
`As Tsc2‘/‘ MEFs failed to down-regulate mTOR activity
`in response to hypoxia, we postulated that Hif levels
`might also be affected. Tsc2‘/‘ MEFS were not impaired
`in the up-regulation of Hif in response to hypoxia. How-
`ever, whereas Tsc2*/* MEFs down-regulated Hif with
`prolonged hypoxia, Hif levels remained elevated in
`Tsc2’/' MEFs (Fig. 1D). Consistent with the idea that
`increased Hif levels in T502‘/‘ cells under prolonged hy-
`poxia result from increased mTOR activity, treatment
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`Figure 1. Tsc2 regulates mTOR in response to hypoxia. (A) Western blot analysis of Tsc2*(* and Tsc2‘(’ MEFs. (Hif) Hif—l(1 and/or
`Hif-2a; (S6K-P) S6K phosphorylated T389; (S6-P) S6 phosphorylated on S235/236. Left panel shows MEF in 0.05% serum or following
`serum addition (10% serum for 45 min) pretreated or not with raparnycin (1.5 h prior to serum addition). Right panel shows MEFS
`exposed to hypoxia for the indicated periods of time. (B) Western blot analysis of Tsc1“/* and Tsc1‘(‘ mouse 3T3 cells treated with
`hypoxia for the indicated periods of time. (C) Western blot analysis of input (left) and 7mGTP—bound (right) proteins from extracts of
`7’sc2*(‘ and Tsc2’(’ MEFs exposed to hypoxia for the indicated periods of time. (D,E) Western blot analysis of extracts from Tsc2*(“ and
`Tsc2’(’ MEFS exposed to hypoxia for the indicated periods of time. In E all the cells were treated with rapamycin for 26 h prior to lysis
`regardless of the duration of hypoxia.
`
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`Brugarolas at al.
`
`with rapamycin restored the down—regulation of Hif nor-
`mally observed after prolonged hypoxia (Fig. 1E).
`The importance of Tsc2 in the regulation of mTOR by
`hypoxia is further supported by reconstitution experi-
`ments. Tsc2"/‘ MEFs were infected with a retrovirus en-
`codingepitope-tagged human Tsc2. Retrovirally trans-
`duced Tsc2'/' MEFS achieved Tsc2 protein levels that
`were similar to endogenous levels in Tsc2*/* MEFS (Fig.
`2A}. Reintroduction of Tsc2 into Tsc2‘/‘ MEFs restored
`the down-regulation of S6 phosphorylation by hypoxia
`(Fig. 2A).
`To determine the generalizability of our observations,
`the role of Tscz in mTOR regulation by hypoxia was
`examined in other cell types. Tsc2 knockdown with two
`different Tsc2 siRNAs in HEK293 and HeLa cervical car-
`cinoma cells blocked the down—regulation of S6 phos-
`phorylation by hypoxia (Fig. 2B; Supplementary Fig. 2.),
`indicating that Tsc2 is required for mTOR regulation by
`hypoxia in multiple cell types.
`
`Tsc2 inactivation confers a proliferative advantage
`under hypoxia
`
`Taken together, these data establish the importance of
`Tscl/Tsc2 in the regulation of mTOR by hypoxia. As
`Tscl/Tsc2 functions as a tumor suppressor, we asked
`whether disruption of this complex would affect cell pro-
`liferation under hypoxic conditions. In keeping with pre-
`vious observations,
`the rates of cell proliferation of
`
`A
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`Figure 2. Tsc2 is both necessary and sufficient for the regula-
`tion of S6 phosphorylation by hypoxia. [A] Western blot analysis
`of T5c2”‘ MEFs retrovirally transduced with either a Tsc2 ex-
`pression vector or an empty vector and treated with hypoxia for
`the indicated periods of time. T552‘/* MEFS are included as con-
`trols. (B) Western blot analysis of HEK293 cells transfected with
`two different synthetic Tsc2 siRNAs (a and b) or a scrambled
`siRNA (Sc) and exposed to hypoxia for the indicated periods of
`time.
`
`2896
`
`GENES 5:. DEVELOPMENT
`
`Tsc2‘/‘ and Tsc2*/* MEFS were very similar under nor-
`moxie conditions for several days (Fig. 3A,- H. Zhang et
`al. 2003). After 4 d in culture the rate of proliferation of
`Tsc2*/* cells began to decline relative to T3132‘/“ cells,
`possibly due to depletion of nutrients and growth factors
`from the media. Consistent with this idea, T5132’/’ but
`not Tsc2*/* MEFs proliferate under conditions of serum
`deprivation (H. Zhang et al. 2003). We next measured the
`proliferation of Tsc2‘/‘ and Tsc2‘/* cells under hypoxic
`conditions. To avoid confounding effects from media
`depletion, the media was changed daily. Under hypoxic
`conditions Tsc.2'/’ MEFs exhibited a marked prolifera-
`tive advantage compared with T.sc2*/* MEFs (Fig. 3B).
`Increased proliferation of T502“/‘ MEFs under hypoxic
`conditions correlated with persistently elevated levels
`of S6 phosphorylation (Fig. 3C) and was abrogated by
`treatment with rapamycin (Fig. 3B). These data suggest
`that failure to down-regulate mTOR in response to hy-
`poxia in Tsc2‘/‘ MEFs confers a growth advantage that
`might contribute to tumor formation in TSC patients. It
`should be noted that the growth inhibitory effect of hy-
`poxia on Tsc2*/* MEFS in these assays is p53 indepen-
`dent since both the Tsc2*/* and Tsc2‘/“ MEFS used here
`lack p53.
`
`.
`
`Tscl /T362 regulation by hypoxia is AMPK
`and Lkbl independent
`
`Energy depletion results in Tscl/Tsc2-mediated mTOR
`inhibition through a mechanism that involves Lkbl and
`AMPK {lnoki et al. 2003b; Corradetti et al. 2004; Shaw et
`al. 2004a}. Since hypoxia might also affect cellular en-
`ergy stores, we sought to determine whether hypoxia
`regulated rnTOR through a similar pathway. AICAR (5-
`arninoomidazole-4-carboxyamide),
`a
`cell-permeable
`AMPK agonist, inhibited mTOR and down-regulated S6
`phosphorylation (Fig. 4A; Kirnura et al. 2003). As previ-
`ously reported (Shaw et al. 2004b}, Tsc2'/‘ cells failed to
`down-regulate S6 phosphorylation in response to AICAR
`despite robust activation of AMPK as determined by
`AMPK phosphorylation in the T loop and the phosphory-
`lation of its substrate acetyl-CoA carboxylase (ACC) (Fig.
`4A), thereby justifying the use of AICAR as an AMPK
`perturbant in the experiments described below.
`To ask whether hypoxia signals are transduccd
`through an energy depletion pathway in our system, we
`first examined the effects of hypoxia on AMPK activity.
`After 4 h of hypoxia, S6 phosphorylation was markedly
`down-regulated without a detectable increase in AMPK
`activity, as measured by ACC phosphorylation (Fig. 4B).
`The ability to detect ACC phosphorylation in response
`to AMPK activation was confirmed by studying cells
`treated with AICAR in parallel (Fig. 4B}. Furthermore, in
`vitro kinase assays with AMPK immunoprecipitates
`from the same lysates used for Figure 4B indicated that
`hypoxia, in contrast to AICAR, did not increase and may
`have decreased AMPK kinase activity (Fig. 4C). These
`results are consistent with a recent study of HEK293
`cells exposed to hypoxia for 30 min (Arsham et al. 2003].
`We next asked whether AMPK activity is necessary for
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`Hypoxia regulation of mTOR by REDD1 and TSCI/TSC2
`
`W
`
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`
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`
`Figure 3. Tsc2 loss confers a proliferative advan-
`tags under hypoxic conditions.
`(A) Proliferation
`rates of Tsc2‘/‘ and Tsc2“(" MEFS under normoxic
`conditions.
`(BI Proliferation rates under hypoxic
`conditions of Tsc2‘/‘ and T3132”/‘ MEFs (treated or
`not with rapamycin). Error bars for A and B equal
`one standard deviation (n = 3]. Note different Y-axis
`scales in A and B.
`(C) Western blot analysis of
`Tsc2*(* and Tsc2"/“ MEFS cultured in parallel under
`hypoxic conditions for the indicated number of days.
`
`‘I25-L124‘-
`’Fsc2+z/+
`3401334
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`the hypoxia—inducecl down-regulation of mTOR func-
`tion. Pharmacological inhibition of AMPK with com-
`pound C (Inoki et al. 2003b) at doses sufficient to block
`the down-regulation of S6 phosphorylation by AICAR
`did not block the down-regulation of S6 phosphorylation
`by hypoxia (Fig. 4D). Thus AMPK is required for AICAR
`signaling but not for hypoxia signaling.
`Next we examined the requirements for the AMPK
`kinase Lkbl. Consistent with previous studies, Lkb1‘/‘
`MEFs failed to down-regulate S6 phosphorylation in re-
`sponse to AICAR (Fig. 4E, Shaw et al. 2004b]. In contrast,
`we found that Lkbl loss did not affect the down-regula-
`tion of S6 phosphorylation by hypoxia (Fig. 4F). in addi-
`tion, HeLa cells, which are defective for Lkbl (Tiainen et
`al. 1999), also down-regulated S6 phosphorylation in re-
`sponse to hypoxia but not to AICAR (Supplementary Fig.
`3) indicating that Lkbl, like AMPK, is dispensable for
`hypoxia signaling. We noted, however, that ACC and
`AMPK do become phosphorylated in HeLa cells after
`prolonged hypoxia, possibly due to a non—Lkbl AMPK
`kinase (Supplementary Fig. 3). Nonetheless, inhibition of
`S6 phosphorylation occurred prior to appreciable AMPK
`activation in these cells, again in keeping with the idea
`that the acute down-regulation of mTOR in response to
`hypoxia is AMPK independent. It is possible that signal-
`ing to Tscl /Tsc2 in response to chronic hypoxia involves
`both Lkbl/AMPK-dependent and independent pathways
`since prolonged hypoxia would predictably lead to ATP
`depletion and accumulation of AMP.
`
`Regulation of mTOR by hypoxia requires
`Reddl induction
`
`To begin to unravel the signaling pathway whereby hy-
`poxia regulates Tscl/Tscl, we asked whether
`this
`mechanism required de novo transcription. Provoca-
`tively, inhibition of transcription with actinomycin D
`blocked the down-regulation of S6 phosphorylation by
`hypoxia, indicating that de novo transcription is neces-
`sary for hypoxia signaling (Fig. 5A). As a control actino-
`rnycin D alone did not affect baseline S6 phosphoryla-
`tion (Fig. 5A).
`
`Recently, the Drosophila ortholog of the hypoxia—in-
`ducible Redd] gene was isolated in a screen for suppres-
`sors of insulin signaling (Reiling and Hafen 2004). Redd]
`was originally identified by several groups as a gene that
`is induced at the mRNA level in response to stresses
`such as hypoxia or DNA damage (Ellisen et al. 2002,-
`Shoshani et al. 2002). Reddl encodes a protein with a
`predicted MW of 25 kDa that lacks any known func-
`tional domains (Ellisen et al. 2002; Shoshani et al. 2002).
`Reddl belongs to a family of highly conserved proteins
`that includes Reddl, which might also be regulated by
`hypoxia (Cuaz-Perolin et al. 2004}. Redd orthologs have
`been shown to inhibit insulin signaling in Drosophila
`and epistasis analysis suggests that they act upstream of
`Tscl/Tscl (Reiling and Hafen 2004).
`To ascertain whether Reddl was involved in the regu-
`lation of the mTOR by hypoxia, we first examined Reddl
`induction in response to hypoxia in MEFs. Consistent
`with previous data, Reddl mRNA levels were induced
`by hypoxia (Fig. 5B,- Shoshani et al. 2002). Redd2 expres-
`sion, however, was not detected in MEFs (data not shown).
`To ask whether Reddl
`is necessary for hypoxia—in—
`duced down—regulation of mTOR function, we obtained
`Reddl‘/‘ MEFS (K. Lei and L.W. Ellisen, in prep). In con-
`trast to Reddl‘/* MEFS, Reddl‘/’ MEFs failed to down-
`regulate phosphorylation of 86K and S6 in response to
`hypoxia (Fig. 5C). These data indicate that Reddl, like
`Tscl and Tsc2,
`is necessary for the hypoxia—induced
`down-regulation of mTOR signaling. This requirement ‘
`was specific because Reddl’/’ MEFS,
`in contrast
`to
`Tsc2‘/‘ MEFS, down—regulated S6 phosphorylation in re-
`sponse to serum deprivation (Fig. 6A,B). These data in-
`dicate that Reddl regulates mTOR signaling only in «re-
`sponse to certain stimuli, such as hypoxia.
`
`Reddl is sufficient to dowrmegulate
`S6K1 phospliorylation
`
`We next asked whether Reddl is sufficient to down-regu-
`late mTOR function. For this purpose,
`the effects of
`Reddl expression on S6K phosphorylation were deter-
`mined. HEK293 cells were transfected with HA-tagged
`
`GENES 8t DEVELOPMENT
`
`2897
`
`Roxane Labs., Inc.
`Exhibit 1019
`
`Page 005
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`Roxane Labs., Inc.
`Exhibit 1019
`Page 005
`
`

`
`Brugamlas et al.
`
`A
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`B
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`smioun
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`inTOR regulation by hypoxia is both AMPK and Lkbl independent. (A) Western blot analysis of extracts of Tsc2*/” and
`Figure 4.
`Tsc2‘/‘ MEFS treated with AICAR for the indicated periods of time. (ACCP) Acetyl-CoA carboxylase phosphorylated at 579; (AMPK—P)
`AMPK phosphorylated at T172. (B) Western blot analysis of Tsc2*/‘ MEFS after 4 h of treatment with either hypoxia or AICAR. (C) In
`vitro AMPK kinase assay of the same extracts used in B irnrnunoprecipitated in antibody excess with a polyclonal anti-AMPK (oz
`AMPK) antibody or normal rabbit IgG (1gG). Samples were normalized for protein concentration prior to immunoprecipitation. Error
`bars equal one standard deviation (:1 = 3). Also shown are background activities of immunoprecipitates incubated in the absence of
`substrate (SAMS peptide). (D) Western blot analysis of Tsc2“”' cells pretreated or not with the AMPK inhibitor compound C and
`exposed to either AICAR or hypoxia for the indicated periods of time. All cells treated with compound C were exposed to the drug for
`9.5 h. (E,F) Western blot of Lkb1*/‘ or LI<b1'/' MEFS treated with AICAR (E) or hypoxia (F) for the indicated periods of time.
`
`human Reddl (R1) or a mutant lacking its evolutionarily
`conserved central region (R1dC,~ amino acids 96-153 out
`of 232). To exclude signal from untransfected cells, and
`thereby increase signal/noise ratio, a plasmid encoding
`HA—S6K1 (SGK) was included in the transfection mix.
`HA—S6Kl was then recovered by anti-HA imInunopre—
`cipitation and immunoblotted with an antibody specific
`for pl1ospho~S6Kl. Wild~type Reddl, but not mutant
`Reddl, down-regulated S6K1 phosphorylation (Fig. 7A).
`As a control, S6Kl phosphorylation was abrogated in
`cells treated with rapaniycin (Fig. 7A). These data indi-
`cate that Reddl
`is sufficient
`to doWn—regulate S6Kl
`phosphorylation and that
`this function requires the
`Reddl central domain.
`‘
`Simultaneous overexpression of two Redd orthologs in
`Drosophila, Scylla and Charybdis, results in additive ef—
`fects with respect to cell size (Reiling and Hafen 2004).
`Since cell size in Drosophila is controlled by 36K, we
`asked whether Redd2 also down-regulated S6KI phos-
`phorylation, For this purpose, we repeated these experi-
`ments with HA—Redd2 (R2), alone or in combination
`with Reddl. Reddl, like Reddl, inhibited S6Kl phos-
`phorylation and the effects of Reddl and Reddl together
`were possibly additive (Fig. 7A). While the same epitope
`tag in Reddl (or 2) and S6Kl, combined with immuno—
`precipitation, could theoretically confound our results,
`qualitatively similar effects were observed when S6Kl
`
`2898
`
`GENES & DEVELOPMENT
`
`phosphorylation was analyzed directly from cell lysates
`(data not shown).
`
`Down-regulation of 86K phospliorylation by Reddl
`requires Tsc2
`
`To ask whether Reddl functions upstream of Tsc2, simi-
`lar assays were performed in HeLa cells in which Tsc2
`function was impaired using siRNA. The effect of Reddl
`and Redd2 on S6Kl phosphorylation in HeLa cells trans-
`fected with irrelevant siRNA (Sc; scrambled) mirrored
`those observed in HEK293 cells (Fig. 7, cf. B and A). In
`stark contrast, the effects of Reddl and Redd2, alone or
`in combination, on S6K1 phosphorylation were corn~
`pletely abrogated in cells treated with either of two dif»
`ferent Tsc2 siRNAs (a and b) (Fig. 7B).
`To evaluate the effects of Reddl on endogenous (rather
`than ectopic) S6K as well as S6 phosphorylation, and its
`dependency on Tscl/Tsc2 for signaling, we generated a
`HA-Reddl-inducible cell line (Fig. 7C). These cells were
`transfected with siRNA (as above; Sc, Tsc2 a and b) and
`subsequently placed in media with tetracycline to in-
`duce I-IA-Reddl. Reddl induction resulted in a profound
`down-regulation of endogenous 56K and S6 phosphory-
`lation unless Tscl was downregulated by siRNA (Fig.
`7C). Therefore downregulation of S6K phosphorylation
`by Reddl and Redd2 requires Tsc2. In addition, S6K
`
`Roxane Labs., Inc.
`Exhibit 1019
`
`Page 006
`
`Roxane Labs., Inc.
`Exhibit 1019
`Page 006
`
`

`
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`Hypoxia regulation of mTOR by REDD1 and TSC1/TSC2
`
`Figure 5. Down-regulation of mTOR function by hypoxia
`requires Reddl. (A) Western blot analysis of Tsc2*/* MEFS
`pretreated (.30 min prior to initiation of the hypoxia time
`course) or not, with Actinorriycin D and exposed to hypoxia
`for the indicated periods of time. Shown in parallel, analysis
`of Tsc2*(* cells treated with Actinomycin D for the indi-
`cated periods of time. Northern blot (B) and Western blot (C)
`analysis of Reddl“ and Reddl‘/‘ MEFS treated with by-
`poxia for the indicated periods of time.
`
`phosphorylation in Reddl’/‘ cells was down-regulated
`by treatment with rapamycin (data not shown), in keep-
`ing with the idea that Reddl acts upstream of mTOR.
`While we cannot formally exclude that Reddl acts in a
`parallel pathway, our data suggest that Reddl acts up-
`stream of
`the Tscl/TscZ complex to down-regulate
`mTOR function in response to hypoxia.
`
`Discussion
`
`The data presented here indicate that the down-regula-
`tion of mTOR function by hypoxia requires Tscl/Tsc2.
`Under conditions of hypoxia, Tsc2-deficient cells fail to
`down-regulate 86K phosphorylation (and activity) and
`accumulate Hif abnormally. Disruption of the Tscl/
`Tsc2 complex results in increased cell proliferation un-
`der hypoxic conditions. Hypoxia signaling requires nei-
`ther AMPK nor Lkbl but does require de novo transcrip-
`tion and the expression of Reddl. Reddl is necessary and
`sufficient for the down-regulation of mTOR by hypoxia.
`Our data reveal that mTOR regulation by hypoxia re-
`quires Tscl/Tsc2 in multiple cell types. Tsc2 was essen-
`tial for hypoxia-induced mTOR down-regulation in three
`
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`is required for growth factor
`Figure 6. Tsc2 but not Reddl
`signaling. Western blot analysis of Tsc2*(* and Tsc2‘/’ MEFs (A)
`or Reddl‘/“ and Redd1"’ MEFs (B) after scrum deprivation for
`the indicated periods of time.
`
`different cell types, MEFS, HEK293 cells, and HeLa cells.
`The requirement of Tsc2 for the down-regulation of
`mTOR in multiple diverse cell
`types suggests that
`mTOR regulation by hypoxia universally requires Tsc2.
`The reg