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NATURE CELL BIOLOGY VOL 4 SEPTEMBER 2002 http://www.nature.com/naturecellbiology658
Akt regulates growth by directlyphosphorylating Tsc2
Christopher J. Potter, Laura G. Pedraza and Tian Xu*Howard Hughes Medical Institute, Department of Genetics, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue,
New Haven, CT 06536-0812, USA
*e-mail: [email protected]
Published online: 2002; DOI:10.1038/ncb840
The direct mechanism by which the serine/threonine kinase Akt (also known as protein kinase B (PKB)) regulatescell growth is unknown. Here, we report that Drosophila melanogaster Akt/PKB stimulates growth by phosphorylat-ing the tuberous sclerosis complex 2 (Tsc2) tumour suppressor and inhibiting formation of a Tsc1–Tsc2 complex. Weshow that Akt/PKB directly phosphorylates Drosophila Tsc2 in vitro at the conserved residues, Ser 924 andThr 1518. Mutation of these sites renders Tsc2 insensitive to Akt/PKB signalling, increasing the stability of theTsc1–Tsc2 complex within the cell. Stimulating Akt/PKB signalling in vivo markedly increases cell growth/size, dis-rupts the Tsc1–Tsc2 complex and disturbs the distinct subcellular localization of Tsc1 and Tsc2. Furthermore, allAkt/PKB growth signals are blocked by expression of a Tsc2 mutant lacking Akt phosphorylation sites. Thus, Tsc2seems to be the critical target of Akt in mediating growth signals for the insulin signalling pathway.
Tuberous sclerosis (TSC) is a relatively common autosomaldominant disorder that occurs in approximately 1 in 6,000births and is characterized by the presence of hamartomas —
disorganized, but differentiated, benign growths — in a wide vari-ety of organs, such as the brain, skin, kidney, lung and heart1. Themost severe manifestations of TSC occur from hamartomas of thebrain that can result in epilepsy, mental retardation and autism1,2.Mutations of two tumour suppressor genes, TSC1 and TSC2, areequally responsible for familial TSC3,4. TSC1 encodes the protein,hamartin (or TSC1), which contains a carboxy-terminal coiled-coiled domain3, whereas TSC2 encodes tuberin (or TSC2), whichcontains an amino-terminal coiled-coil domain and a C-terminalRab guanosine trisphosphatase activation protein (GAP)-likedomain4,5.
The clinical manifestations of mutations in TSC1 and TSC2 arevirtually indistinguishable, suggesting that the two proteins functionin the same biological process6. The presence of coiled-coil domains,which are implicated as sites of protein–protein interactions7, with-in the two proteins further raised the possibility that they mightform a complex in vivo8. Indeed, the coiled-coil domains of TSC1and TSC2 bind each other in yeast two-hybrid analysis8, and TSC1and TSC2 co-immunoprecipitate in vivo9–11. However, the function-al significance of TSC1 and TSC2 association and the biologicalpathway in which TSC proteins function remains unclear.
In Drosophila, genetic screens of mosaic animals have been usedto identify genes that, when mutated, result in overgrowth of theclonal population of mutant cells12–21. Three classes of overgrowthmutants have been genetically identified from such screens. First,mutations of genes, such as lats, cause over-proliferation of mutantcells, which develop into tumours12. Second, mutations of genes,such as slimb, result in increased cell proliferation as a consequenceof duplicated structures13. Third, mutant cells acquire a growthadvantage and exhibit an increase in cell size. This class includesmutant alleles of Drosophila Tsc1 (Tsc1)18–20 and Drosophila Tsc2(Tsc2)14, as well as mutant alleles of the Drosophila homologue ofthe tumour suppressor PTEN15,17,22. Furthermore, ectopic overex-pression of Tsc1 or Tsc2 alone in Drosophila tissues has no effect,but co-overexpression of Tsc1 and Tsc2 results in marked decreasesin cell growth and proliferation18–20. As Tsc1 and Tsc2 form a com-plex in vitro18 and in vivo20, these results indicate that Tsc1 and Tsc2
function in vivo as a complex to regulate cellular growth.The similarity in phenotypes caused by mutation of Drosophila
Pten and Tsc1 or Tsc2 suggested that they might share a commonbiological function18. Indeed, patients with hereditary mutations ofhuman PTEN, such as TSC1 and TSC2 patients, also develophamartomas in multiple organs. PTEN functions to antagonize theconserved insulin/phosphatidylinositol-3-OH kinase (PI(3)K) sig-nalling pathway in humans and flies15,17,22,23. PTEN is also one of themost frequently mutated genes involved in the development ofhuman cancers24, indicating that the insulin signalling pathway ispivotal in tumorigenesis. As TSC1 and TSC2 also function astumour suppressors, these results suggested that TSC1 and TSC2
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Figure 1 Akt alters the localization of Tsc1 and Tsc2. a–c, Tsc1 (anti-Myc, red)and Tsc2 (anti-Flag, green) colocalize in the cytoplasm when expressed together inthe posterior region of the eye imaginal disc. d–f, Expression of Dakt1 results in adiffuse cytoplasmic localization of Tsc1 and Tsc2. Dakt1 expression also inducesan increase in cell size. Scale bars represent 2.5 µm. The genotypes used are list-ed in the Methods.
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might also function as antagonists of the insulin signalling pathway.In Drosophila, mutations of positive components of the insulin sig-nalling pathway — insulin receptor (IR), PI(3)K, Akt/PKB and S6kinase (S6K) — resulted in marked decreases in cell size25–28. To deter-mine if, and where, Tsc1 and Tsc2 function within the insulin sig-nalling pathway, classic genetic epistasis experiments were performed.
The genetic data showed that the Tsc1–Tsc2 complex functionsbetween Akt and S6K in the insulin signalling pathway18. However,the molecular mechanism by which the insulin/PI(3)K/Akt path-way transduces signals through Tsc1–Tsc2, and how Akt regulatesTsc1–Tsc2 activity, is unknown.
The serine/threonine kinase Akt is pivotal in multiple process-es29–31. Akt regulates cell growth through the indirect activation ofTOR (target of rapamycin) and S6K. Indeed, expression of Akt inDrosophila and mammalian tissues results in marked increases incell growth/size32,33. Akt is also pivotal in tumorigenesis, as the abil-ity of PTEN to function as a tumour suppressor results from itsfunction in negatively regulating the activity of Akt34. Mutations ofPTEN result in activation of the Akt kinase, and the ability of Aktto stimulate growth is essential for its oncogenic activity35.However, the direct mechanism by which Akt stimulates growth invivo is unclear. Using Drosophila as a model, we report here that Aktstimulates growth by phosphorylating Tsc2 and inhibiting forma-tion of the Tsc1–Tsc2 complex.
ResultsAkt disrupts the subcellular localization of Tsc1 and Tsc2. Toinvestigate the relationship between the Tsc1–Tsc2 complex andAkt signalling, we examined the effects of Akt expression on thelocalization of Tsc1 and Tsc2. If the Tsc1–Tsc2 complex does indeedfunction downstream of Akt, then the subcellular localization ofTsc1–Tsc2 could be affected by Akt signalling. As previouslydemonstrated with mammalian Tsc1 and Tsc2 (refs 8–10),Drosophila Tsc1 and Tsc2 colocalized in the cytoplasm in a punctate
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Figure 2 Insulin/Akt inhibits the stability of the Tsc1–Tsc2 complex. a, Aschematic representation of the Drosophila and human Tsc2 protein structure show-ing the putative Akt phosphorylation sites. Only the conserved Akt phosphorylationsites in human Tsc2 are shown. A sequence comparison of the four DrosophilaTsc2 Akt phosphorylation sites is also shown. The asterisk indicates the predictedphosphorylated residue. cc, coiled-coil. b, Insulin inhibits the association of Tsc2with Tsc1. Extracts from transfected S2 cells were immunoprecipitated for Tsc2and western blotted for Tsc1. Tsc2 co-immunoprecipitates Tsc1 when both aretransfected (lane 1). Treatment of S2 cells with insulin decreases the association ofTsc2 with Tsc1 (lane 2). This decrease in stability of the Tsc1–Tsc2 complex can be
blocked by pre-treatment of S2 cells with wortmannin (lane 3). Cotransfection ofTsc1 with Tsc2∆Akt-P immunoprecipitates Tsc1 to a similar degree as wild-type Tsc2(lane 4). However, this Tsc1–Tsc2∆Akt-P complex is not disrupted by insulin treatment(lane 5). c, Akt expression decreases the stability of the Tsc1–Tsc2 complex.Transfection of constitutively active Akt32 (Akt) reduces co-immunoprecipitation ofTsc1 by Tsc2 (lane 4). Transfection of Tsc2∆Akt-P stabilizes the Tsc1–Tsc2∆Akt-P com-plex in the presence of Akt (lane 6). d, Akt expression increases serine/threoninephosphorylation of Tsc2 in vivo. Larvae were untreated (−) or heat-shocked (+) toinduce ubiquitous expression of Dakt1. Extracts were immunoprecipitated for Tsc2and western blotted for phospho-serine/threonine residues.
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Figure 3 Akt phosphorylates Tsc2 at Ser 924 and Thr 1518 in vitro.Recombinant Tsc1 or Tsc2 were incubated with active (+) or heat-inactivated (−)recombinant human Akt1 in the presence of 32P-ATP. The upper panel is an autora-diograph of 32P incorporation into Tsc2. The lower panel shows the Coomassiestaining of the recombinant Tsc1 and Tsc2 proteins.
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pattern (Fig. 1a–c). However, Akt expression disrupted the distinctsubcellular colocalization of Tsc1 and Tsc2 (Fig. 1d–f). Theseresults suggest that Akt can directly inhibit formation of theTsc1–Tsc2 complex in vivo.The Tsc1–Tsc2 complex is disrupted by insulin signalling. To exam-ine the effect of insulin/PI(3)K/Akt signalling on the Tsc1–Tsc2complex in greater detail, we performed co-immunoprecipitationexperiments in Drosophila S2 cells. Confirming the formation of anin vitro and in vivo complex18,20, cotransfected Tsc1 and Tsc2 werefound to co-immunoprecipitate (Fig. 2b,c). Treatment of S2 cellswith insulin resulted in a decrease in the co-immunoprecipitation ofTsc1 by Tsc2 (Fig. 2b, lane 2), which could be suppressed by pre-treatment of the cells with the PI(3)K inhibitor wortmannin (Fig. 2b,lane 3). A possible mechanism through which insulin/PI(3)K/Aktcould disrupt the interaction of Tsc1 and Tsc2 might be by directphosphorylation of Tsc1 or Tsc2. We therefore examined the proteinsequences of Tsc1 and Tsc2 for the Akt phosphorylation consensusmotif, Arg-X-Arg-X-X-Ser/Thr, where X is any amino acid6
(Fig. 2a). Tsc2 contains four potential Akt phosphorylation sites,two of which (Ser 924 and Thr 1518) are conserved in all reportedTsc2 proteins, suggesting that these residues could be phosphory-lated by Akt (Fig. 2a and data not shown). By contrast, Tsc1 con-tains one potential Akt phosphorylation site that is not found inother Tsc1 homologues (data not shown). Interestingly, mutationof all potential Akt phosphorylation sites on Tsc2 (Tsc2∆Akt-P) result-ed in increased stability of the Tsc1–Tsc2∆Akt-P complex in the pres-ence of insulin (Fig. 2b, lane 5). These results suggest that theTsc1–Tsc2 complex can be disrupted by insulin/PI(3)K signalling,perhaps through activation of Akt. To examine this in greaterdetail, constitutively active myristoylated Akt32 was cotransfectedwith Tsc1 and Tsc2 (Fig. 2c, lane 4). This resulted in a markeddecrease in the co-immunoprecipitation of Tsc1 by Tsc2. By con-trast, the Tsc1–Tsc2∆Akt-P complex was maintained after inductionof active Akt (Fig. 2c, lane 6). These results suggest that phospho-rylation of the conserved Akt phosphorylation sites on Tsc2inhibits its binding to Tsc1.
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Figure 4 Mutation of Ser 924 and Thr 1518 in Tsc2 results in an activatedTsc2 protein. a, SEM of a control adult eye. b, Overexpression of Tsc1 with Tsc2in the proliferating cells of the eye disc results in a 31% decrease in ommatidianumbers. c,d, Expression of Tsc2 mutated at Ser 924 (c) or Thr 1518 (d) results ina greater decrease in ommatidia numbers. e,g, Expression of Tsc2 mutated at Ser924 and Thr 1518 (e), or ∆Akt-P (g) results in a 75% decrease in ommatidia num-bers. f, Expression of Tsc2 mutated at Thr 437 and Thr 1054 affects ommatidia
numbers similarly to expression of wild-type Tsc2. h, Overexpression of Tsc2∆Akt-P inproliferating cells (without expression of Tsc1) also reduces ommatidia numbers by9%. i, A histogram quantifying ommatidia number from a–h. Results are means ±standard deviation. At least ten eyes were examined for each genotype. All eyesare from females. Scale bars represent 50 µm. The genotypes used are listed inthe Methods.
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Akt phosphorylates Tsc2 in vitro and in vivo. To determine if Aktcould induce phosphorylation of Tsc2 in vivo, we immunoprecipi-tated Tsc2 from larvae that ubiquitously expressed Drosophila Aktafter induction of heat shock (Fig. 2d; see Methods). Indeed,expression of Akt resulted in increased reactivity of Tsc2 to anti-phospho-serine/threonine antibodies (Fig. 2d). To determine if Aktcould phosphorylate Tsc2 directly, we used an active recombinanthuman Akt in kinase assays with recombinant Tsc1 and Tsc2 pro-teins (Fig. 3). Phosphorylation of Akt was indicated by incorpora-tion of 32P. No obvious phosphorylation of Tsc1 was detected(Fig. 3, lane 1), whereas Tsc2 showed robust phosphorylation(Fig. 3, lane 3). Furthermore, mutation of Tsc2 at Ser 924 or Thr1518 reduced incorporation of 32P (Fig. 3, lanes 4 and 5), whereasincorporation was completely abolished by mutating both con-served sites (Fig. 3, lane 6). By contrast, 32P incorporation occurrednormally after mutation of Tsc2 at the non-conserved sites, Thr 437and Thr 1054 (Fig. 3, lane 7). As with Tsc2S924A/T1518A, mutation of allfour Tsc2 Akt phosphorylation sites (∆Akt-P) completely abolished32P incorporation (Fig. 3, lane 8). These results indicate that Akt
can directly phosphorylate Tsc2 on the conserved Ser 924 andThr 1518 residues.Mutation of the Akt phosphorylation sites on Tsc2 stabilizes andactivates Tsc1–Tsc2. Co-expression experiments with Tsc1 and Tsc2suggest that Tsc1 and Tsc2 function as an active complex18–20. As theTsc2 Akt-phosphorylation-site mutants generate a more stableTsc1–Tsc2 complex, expression of these Tsc2 mutants should resultin increased TSC-related activity in vivo. To test this hypothesis, wegenerated transgenic animals that express the Tsc2 Akt-phosphory-lation-site mutants and used the UAS/GAL4 binary system ofectopic expression37. To assess the activity of the Tsc1–Tsc2 com-plex, the genes were expressed in the proliferating cells of the eyeimaginal disc. Expression of wild-type Tsc2 and Tsc1 in these cellsresults in a decrease in cell proliferation, as demonstrated by a 31%decrease in ommatidia numbers in the adult fly18–20(Fig. 4b,i).Overexpression of Tsc1 with Tsc2S924A or Tsc2T1518A resulted in a morepronounced decrease in ommatidia numbers (50%; Fig. 4c,d,i).Expression of Tsc1 with the double mutant Tsc2S924A/T1518A resulted inan even more marked decrease in ommatidia numbers (75%;
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Figure 5 Mutation of Ser 924 and Thr 1518 in Tsc2 inhibits Akt-stimulatedgrowth. a, SEM of a control adult eye. b, Overexpression of Dakt1 in the differenti-ating cells of the eye disc results in a 150% increase in ommatidia size32. c,d, Thisincrease in cell growth is not affected by expression of Tsc1 (c) or Tsc2 (d) alone.e, Expression of Tsc1 and Tsc2 with Dakt1 partially suppresses the enlargedommatidia phenotype. f, The Dakt1 overexpression phenotype is fully suppressedby expression of Tsc1 and Tsc2∆Akt-P. In this case, the ommatidia are 14% smallerthan the control. g,h, Expression of the Tsc2S924A (g) or Tsc2T1518A (h) mutants par-tially suppress the increase in ommatidia size caused by Dakt1. i, Expression ofTsc2S924A/T1518A completely suppresses the Dakt1 overexpression phenotype. j, By
contrast, overexpression of Tsc2T437A/T1054A has no effect. k, Expression of Tsc2∆Akt-P,similarly to expression of Tsc2S924A/T1518A, also fully suppresses the Dakt1 pheno-type. l, Overexpression of Tsc2∆Akt-P alone (without Dakt1) results in a similar 15%decrease in ommatidia size. m, A histogram of the percentage change in omma-tidia size from a–l. Results are means ± standard deviation and expressed as a per-centage of wild-type ommatidia size. At least 12 eyes were examined for eachgenotype. All eyes are from females. Higher magnifications are shown in the lowerpanels. Scale bars represent 60 µm in the upper panels and 10 µm in the lowerpanels. The genotypes used are listed in the Methods.
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Fig. 4e,i), comparable with the expression of Tsc2∆Akt-P (Fig. 4g,i).By contrast, expression of Tsc2 mutated at residues Thr 437 andThr 1054 did not alter the in vivo activity of Tsc2 (Fig. 4f,i). Becauseexpression of Tsc1 or Tsc2 alone has no obvious effect18–20, it wassurprising that overexpression of Tsc2∆Akt-P alone resulted in a slightdecrease in ommatidia numbers (9%; Fig. 4h,i). One explanation isthat Tsc2∆Akt-P binds to endogenous Tsc1, resulting in a more stableTsc1–Tsc2 complex. Together, these results indicate that productionof a more stable Tsc1–Tsc2 complex also results in increased activ-ity in vivo.Akt mediates growth through Tsc2 in vivo. In Drosophila, the mostprominent phenotype caused by mutations of Tsc1 or Tsc2 is anincrease in cell growth14,18–20. Similarly, alterations of Akt signallingalso result in profound effects on growth32,38. We therefore ques-tioned whether the Tsc1–Tsc2 complex could affect Akt-mediatedgrowth. Ectopic expression of Akt in the differentiating cells of theeye disc results in marked increases in cell growth, as demonstratedby a 154% increase in ommatidia size32 (compare Fig. 5a and b).Expression of either Tsc1 or Tsc2 alone had no effect on Akt-medi-ated growth (Fig. 5c,d,m). Co-expression of Tsc1 and Tsc2 partiallysuppressed the Akt overexpression phenotype, but ommatidia sizedid not return to control levels (Fig. 5e,m). The most probableexplanation is that the ectopically expressed Tsc1–Tsc2 complexwas still readily inhibited by the co-expression of Akt. This suggest-ed that removal of the Akt phosphorylation sites on Tsc2 shouldmake the Tsc1–Tsc2 complex refractory to Akt signalling in vivo.
Indeed, expression of Tsc1 with Tsc2∆Akt-P completely suppressed theAkt-mediated growth and even resulted in a 14% decrease inommatidia size (Fig. 5f,m). Strikingly, expression of Tsc2∆Akt-P alone(without co-expression of Tsc1) could also completely suppressAkt-mediated cell growth (Fig. 5k,l,m). We therefore examined fur-ther the contributions of the individual Akt-phosphorylation sitesin mediating Akt-induced growth. Expression of Tsc2S924A orTsc2T1518A resulted in a partial suppression of Akt-mediated growth(Fig. 5g,h,m). Furthermore, expression of Tsc2S924A/T1518A completelysuppressed Akt-mediated cell growth (Fig. 5i,m). However, expres-sion of Tsc2T437A/T1054A had little effect (Fig. 5j,m). These data suggestthat most, if not all, Akt-mediated growth signals are transduced byphosphorylation of Tsc2 at Ser 924 and Thr 1518. Phosphorylationat Ser 924 and Thr 1518 of Tsc2 would probably then disrupt theTsc1–Tsc2 complex. To examine whether most Akt-mediatedgrowth is indeed transduced by the Tsc1–Tsc2 complex, Tsc1mutant clones were induced in eyes expressing Akt (seeSupplementary Information, Fig. S1). If Akt mediates growth bynegatively regulating the stability of the Tsc1–Tsc2 complex, thenthe increase in growth caused by disruption of the complex (forexample, mutation of Tsc1) should not be enhanced by ectopicexpression of Akt. Indeed, increased expression of Akt did notincrease the size of Tsc1 mutant ommatidia (compareSupplementary Information, Fig. S1b and c).
To examine whether Tsc2 could affect Akt-mediated growth intissues other than the eye, we generated marked overexpressionclones in the anterior margin of the wing using the ‘FLIP-out’ tech-nique39,40. By this strategy, clones that overexpress the transgene(s)appear yellow. As the wing-bristle/shaft arises from a single cell, thesize of the bristle is a good measure of cell growth. Unsurprisingly,overexpression of Akt resulted in a marked 186% increase in bristlesize (Fig. 6b,j). Conversely, overexpression of Tsc1 and Tsc2 resultedin a 35% decrease in bristle size (Fig. 6c,j) and partial suppressionof the Akt-overexpression bristle phenotype (Fig. 6d,j). By contrast,overexpression of Tsc1 and Tsc2∆Akt-P resulted in complete suppres-sion of the Akt-mediated bristle phenotype (Fig. 6e,j). In addition,
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Figure 6 Akt-stimulated growth in the wing can be abolished by Tsc2∆∆Akt-P.The FLIP-out technique39,40 was used to overexpress marked clones in the anteriormargin of the wing. Bristles that overexpress the genes listed (arrows) are in yel-low. a, A light micrograph of a control wing, showing that bristle size is not signifi-cantly affected by this technique. b, Overexpression of Dakt1 increases bristle sizeby 180%. c, Overexpression of Tsc1 with Tsc2 decreases bristle size by 40%. d, Expression of Dakt1 with Tsc1 and Tsc2 slightly suppresses the Akt overexpres-sion phenotype. e, Expression of Dakt1 with Tsc1 and Tsc2∆Akt-P reduces bristle sizemarkedly. f, Similarly, overexpression of Tsc1 and Tsc2∆Akt-P decreases bristle size.g, Expression of Tsc2 does not affect the Dakt1 overexpression phenotype. h, Expression of Tsc2∆Akt-P with Dakt1 generates smaller bristles. i, Bristle size issimilarly reduced by overexpression of Tsc2∆Akt–P alone. j, A histogram of the per-centage change in bristle size from a–l. Results are means ± standard deviationand expressed as a percentage of bristle size of neighbouring wild-type clones. Atleast 25 bristles were examined for each genotype. Scale bar represents 25 µm.The genotypes used are listed in the Methods.
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Figure 7 S6K suppresses Tsc1–Tsc2∆∆Akt-P-mediated growth reduction.a, Overexpression of Tsc1 and Tsc2∆Akt-P in the differentiating cells of the eye discresults in a marked decrease in ommatidia size. b, Expression of Drosophila S6Kslightly suppresses Tsc1–Tsc2∆Akt-P expression. c, Expression of human S6KD4,which mimics a mitogen-induced phosphorylation state of S6K46, markedly sup-presses Tsc1–Tsc2∆Akt-P-mediated growth inhibition. Inset values represent percent-age of control ± standard deviations in ommatidia size. At least five eyes per geno-type were analysed. All eyes are from females. Higher magnifications are shown inthe lower panels. Scale bars represent 60 µm in upper panels and 10 µm in lowerpanels. The genotypes used are listed in the Methods.
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the bristles were greatly reduced in size, similar to the reductioncaused by expression of Tsc1 and Tsc2∆Akt-P (Fig. 6f,j). Strikingly,expression of Tsc2∆Akt-P alone could also completely suppress theAkt-mediated bristle phenotype (Fig. 6h,j), whereas expression ofwild-type Tsc2 had no effect (Fig. 6g,j). Furthermore, expression ofAkt with Tsc2∆Akt-P also resulted in a 32% decrease in bristle size,similar to bristles that expressed Tsc2∆Akt-P alone (Fig. 6i,j). Theseresults are consistent with the observations in the eye and indicatethat Akt regulates growth through Tsc2.
A potential downstream component of Tsc1–Tsc2 signalling isS6K18. In support of this model, genetic data demonstrated thatcells mutant for both S6K and Tsc2 exhibit the decreased growthmutant phenotype of S6K18. This suggests that S6K function isrequired for Tsc2-mediated growth. Furthermore, unlike expres-sion of Akt, expression of Drosophila or human S6K can partiallysuppress the growth defects caused by overexpression of Tsc1 andTsc2∆Akt-P (Fig. 7). For example, expression of human p70S6K1
increased the size of ommatidia expressing Tsc1 and Tsc2∆Akt-P by40% (Fig. 7c). This lends support to the model in which S6K func-tions downstream of the Tsc1–Tsc2 complex (Fig. 8)18.
DiscussionGenetic experiments previously demonstrated that there is anantagonistic relationship between Tsc1–Tsc2 and insulin/PI(3)K/Aktsignalling18,20. Genetic epistasis experiments showed that cellsmutant for both Akt and Tsc1 (or Tsc2) exhibit an increased growthphenotype that is indistinguishable from those of Tsc1 or Tsc2
mutant cells18,20. By contrast, cells mutant for Tsc and S6K exhibitonly the S6K decreased-cell-size phenotype18. As the double mutantphenotypes are not a combination of the respective single mutantphenotypes, these genetic results indicated that the Tsc1–Tsc2 com-plex functions between Akt and S6K in the insulin signalling path-way18. Furthermore, they do not support the hypothesis that theTsc1–Tsc2 complex functions in a parallel Akt-independent path-way that exerts its effects on growth by converging with the insulinsignalling pathway20.
The genetic epistasis results showing that Tsc1–Tsc2 functionsdownstream of Akt in the insulin signalling pathway suggest thatthe Tsc1–Tsc2 complex could be directly regulated by Akt18.Consistently, this study indicates that the Tsc1–Tsc2 complex func-tions as an integral component of the insulin/PI(3)K/Akt signallingpathway. We have shown that Akt regulates growth by directlyphosphorylating Tsc2, and that all Akt-mediated growth signalsin vivo can be blocked by mutating the conserved Akt phosphory-lation sites in Tsc2. Together, these results indicate that Tsc2 is thecritical target for Akt in the stimulation of growth in Drosophila.
The molecular mechanism through which the Tsc1–Tsc2 com-plex regulates growth is unknown, but it probably involves activa-tion of S6K18 (Fig. 8), as suggested by genetic epistasis data18
(Fig. 7). However, as S6K must be phosphorylated for activation,and neither Tsc1 nor Tsc2 contain a kinase domain, the inhibitionof S6K by the Tsc1–Tsc2 complex is probably indirect. A possiblemediator of Tsc1–Tsc2 signalling is TOR, a protein that directlyphosphorylates S6K in vitro41,42. This suggests that the Tsc1–Tsc2complex might inhibit TOR activity, perhaps by binding to TORand maintaining it in an inactive state. Signalling from Akt woulddisrupt the Tsc1–Tsc2 complex, releasing TOR, and thereby stimu-late S6K activity. Indeed, genetic epistasis experiments betweenDrosophila TOR and Tsc1 indicate that TOR functions epistaticallyto Tsc1 (C.J.P. and T.X., unpublished observations), and thereforedownstream of the Tsc1–Tsc2 complex (Fig. 8).
Our data also suggest a molecular mechanism through whichAkt (and the insulin signalling pathway) regulates Tsc1–Tsc2 activ-ity: direct phosphorylation of Tsc2 by Akt affects the stability, andthus the in vivo activity, of the Tsc1–Tsc2 complex. Indeed, a Tsc2mutant that lacks its conserved Akt phosphorylation sites, and thusforms a more stable complex with Tsc1, functions as a dominant-active molecule in vivo. Because the Akt, Tsc1 and Tsc2 molecules,as well as the Akt phosphorylation sites in Drosophila Tsc2, are con-served from insects to humans, phosphorylation of Tsc2 by Aktmay function as a conserved mechanism of growth control. Thissuggests that the tumorigenic effects of PTEN and Akt are probablycaused in part by their ability to regulate the Tsc1 and Tsc2 tumoursuppressors. Indeed, reagents such as dominant-active Tsc2mutants — molecules that alter Tsc2 phosphorylation or affect thestability of the Tsc1–Tsc2 complex — may provide effective treat-ment for diseases related to the insulin/PI(3)K/Akt/PTEN/TSCpathway.
MethodsPlasmids and site-directed mutagenesisT437A, S924A, T1054A and T1518A missense mutations were introduced into the pBS-Tsc2 con-
struct14 using the QuickChange site directed mutagenesis kit (Stratagene, La Jolla, CA), and mutations
were verified by sequencing before cloning into pUAST. An N-terminal Flag epitope was added by PCR
to generate UAS-Flag-Tsc2, which was confirmed by DNA sequencing. Flag-Tsc2 was cloned into the
metallothionein promoter vector43 to generate pRmHa-3-Flag-Tsc2. To generate pRmHa-3-Flag-
Tsc2∆Akt-P, the DraIII–XhoI fragment from the pBS-Tsc2∆Akt-P construct was shuffled into pRmHa-3-
Flag-Tsc2.
A 6×Myc tag from Atro-6×Myc (S.Z. and T.X., unpublished observations) was added to the C ter-
minus of Tsc1 (ref. 18) by PCR to generate UAS-Tsc1-Myc. The Tsc1-Myc insert was cloned into
pRmHa-3 to generate pRmHa-3-Tsc1-Myc.
A Tsc2 PCR product was cloned into pGEX4T-1 (Pharmacia Biotech, Piscataway, NJ) to generate
the GST–Tsc2 fusion constructs. GST fusion proteins were purified according to the manufacturer’s
protocol. GST–Tsc1 was used as previously described18. Protein concentrations were determined by
Coomassie blue staining of gels containing BSA standards and aliquots of GST–Tsc1–Tsc2. pCaSper-
hs-myr-Dakt1 (ref. 32) was a gift from M. Birnbaum.
IR
PI(3)K
PTEN
Active Inactive
TOR
S6K
Cell growth
Tsc2 Tsc2Tsc1
Tsc1
Akt/PKB P
P
Figure 8 Akt regulates growth by phosphorylating and inhibiting theTsc1–Tsc2 complex. A schematic representation of this work and previous genet-ic epistasis experiments18 illustrates that stimulation of Akt activity, either by activa-tion of IR, PI(3)K or loss of PTEN, results in direct phosphorylation of Tsc2. Thisevent disrupts binding to Tsc1 and thereby inactivates the Tsc complex. Inactivationof the Tsc1–Tsc2 complex results in increased cell growth, possibly mediated bydownstream inhibition of TOR and activation of S6K18.
© 2003 Nature Publishing Group
articles
NATURE CELL BIOLOGY VOL 4 SEPTEMBER 2002 http://www.nature.com/naturecellbiology664
GeneticsUAS-Tsc1 (ref. 18), UAS-Tsc2 (ref. 18), UAS-Dakt1 (ref. 32), ey-GAL4 (ref. 44), pGMR-GAL4 (ref. 45),
Tsc1Q600X (ref. 18), UAS-dS6K (ref. 46) and UAS-p70S6K1 (ref. 46) have been described. Samples for scan-
ning electron microscopy and adult wings were prepared as described18. The ‘FLP-out’ method39,40 was
used for clonal overexpression in the anterior wing margin. Clones were induced in 72-h larvae by 1-h
heat shock at 38 °C. Transgenic animals were generated as described18. At least five different transgenic
lines were tested for each construct.
The genotypes used were as follows: Fig. 1a–c, yw; pGMR-GAL4/UAS-Tsc1-Myc; UAS-Flag-Tsc2/+;
Fig. 1d–f, yw; UAS-Dakt1, pGMR-GAL4/UAS-Tsc1-Myc; UAS-Flag-Tsc2/+.
Fig. 4a, UAS-Tsc2∆Akt-P/+; UAS-Tsc1/+ (control); Fig. 4b, ey-GAL4/UAS-Tsc2; UAS-Tsc1/+; Fig. 4c, ey-
GAL4/UAS-Tsc2S924A; UAS-Tsc1/+; Fig. 4d, ey-GAL4/UAS-Tsc2T1518; UAS-Tsc1/+; Fig. 4e, ey-GAL4/UAS-
Tsc2S924A/T1518A; UAS-Tsc1/+; Fig. 4f, ey-GAL4/UAS-Tsc2T437A/T1054A; UAS-Tsc1/+; Fig. 4g, ey-GAL4/UAS-
Tsc2∆Akt-P; UAS-Tsc1/+; Fig. 4h, ey-GAL4/UAS-Tsc2∆Akt-P.
Fig. 5a, pGMR-GAL4/CyO (control); Fig. 5b, UAS-Dakt1, pGMR-GAL4/CyO; Fig. 5c, UAS-Dakt1,
pGMR-GAL4/+; UAS-Tsc1/+; Fig. 5d, UAS-Dakt1, pGMR-GAL4/UAS-Tsc2; Fig. 5e, UAS-Dakt1, pGMR-
GAL4/UAS-Tsc2; UAS-Tsc1/+; Fig. 5f, UAS-Dakt1, pGMR-GAL4/UAS-Tsc2∆Akt-P; UAS-Tsc1/+; Fig. 5g,
UAS-Dakt1, pGMR-GAL4/UAS-Tsc2S924A; Fig. 5h, UAS-Dakt1, pGMR-GAL4/UAS-Tsc2T1518A; Fig. 5i,
UAS-Dakt1, pGMR-GAL4/UAS-Tsc2S924A/T1518A; Fig. 5j, UAS-Dakt1, pGMR-GAL4/UAS-Tsc2T437A/T1053A;
Fig. 5k, UAS-Dakt1, pGMR-GAL4/UAS-Tsc2∆Akt-P; Fig. 5l, pGMR-GAL4/UAS-Tsc2∆Akt-P.
Fig. 6a, yw hs-FLP; actin>y+>GAL4/+; Fig. 6b, yw hs-FLP; actin>y+>GAL4/UAS-Dakt1; Fig. 6c, yw
hs-FLP; actin>y+>GALl4/UAS-Tsc2; UAS-Tsc1/+; Fig. 6d, yw hs-FLP; actin>y+>GAL4, UAS-
Dakt1/UAS-Tsc2; UAS-Tsc1; Fig. 6e, yw hs-FLP; actin>y+>GAL4, UAS-Dakt1/UAS-Tsc2∆Akt–P; UAS-
Tsc1/+; Fig. 6f, yw hs-FLP; actin>y+>GAL4 /UAS-Tsc2∆Akt–P; UAS-Tsc1/+; Fig. 6g, yw hs-FLP;
actin>y+>GAL4, UAS-Dakt1/UAS-Tsc2; Fig. 6h, yw hs-FLP; actin>y+>GAL4, UAS-Dakt1/UAS-
Tsc2∆Akt–P; Fig. 6i, yw hs-FLP; actin>y+>GAL4/UAS-Tsc2∆Akt–P.
Fig. 7a, UAS-Tsc1/+; pGMR-GAL4/UAS-Tsc2∆Akt-P; Fig. 7b, UAS-Tsc1/UAS-dS6K; pGMR-GAL4/UAS-
Tsc2∆Akt-P Fig. 7c, UAS-Tsc1/UAS-p70S6K1; pGMR-GAL4/UAS-Tsc2∆Akt-P.
ImmunohistochemistryAnimals of genotype yw; UAS-Dakt, pGMR-GAL4 were crossed to UAS-Tsc1-Myc; UAS-Flag-Tsc2 ani-
mals and eye discs dissected from wandering third-instar larvae. The following primary antibodies and
dilutions were used: 3F10 rat anti-haemagglutinin (HA) (1:50; Roche, Nutley, NJ), M2 mouse anti-Flag
(1:35; Sigma, St Louis, MO) and A14 rabbit anti-Myc (1:200; Santa Cruz Biotechnology, Santa Cruz,
CA). Secondary antibodies used were Cy5-conjugated anti-rat, FITC-conjugated anti-mouse and
Rhodamine-Red-conjugated anti-rabbit (1:500; Jackson ImmunoResearch, West Grove, PA). Confocal
images were collected using a BioRad MRC-1024 microscope (Biorad, Hercules, CA).
Cell culture and immunoprecipitationS2 cell culture conditions and induction of Flag–Tsc2 and Tsc1–Myc by a 12-h incubation with
0.7 mM copper sulphate were as described47, except that CellFECTIN (Invitrogen, Carlsbad, CA) was
used to transfect 10 µg total DNA. S2 cells were washed in PBS and lysed for 45 min in RIPA buffer
(50 mM Tris-HCl at pH 7.4, 1% NP-40, 0.5% sodium-deoxycholate, 150 mM sodium chloride, 1 mM
EDTA, 1 mM phenyl methylsulphonyl fluoride and 1 µg ml−1 each of antipain, leupeptin, chymostatin
and pepstatin). Lysates were then incubated with protein G–agarose beads (Roche) pre-bound to anti-
Flag antibodies for 2 h at 4 °C. The immunoprecipitate was washed five times with RIPA buffer and
resolved on 6% SDS-polyacrylamide gel electrophoresis (PAGE) gels. Western blots were performed
with mouse monoclonal anti-Myc (1:5000; Invitrogen) or mouse anti-gigas (1:20, monoclonal
3H1D8F5; Developmental Studies Hybridoma Bank, Iowa City, IA) antibodies. For insulin treatment,
cells were serum-starved for 6 h and incubated with 10 µg ml−1 insulin (Sigma) in serum-free media
for 20 min. In other experiments, cells were pre-treated with 1 µM wortmannin for 20 min before
addition of insulin. Expression of Dakt1 was induced by incubation at 37 °C for 45 min before a 1-h
recovery at 25 °C. Cells not treated with insulin were incubated with 10 µM LY294002 (Calbiochem,
La Jolla, CA) for 1 h before harvesting.
For larval extracts, 25 third-instar larvae of genotype yw, hsFLP; UAS-Dakt1, actin>y+>GAL4 were
incubated for 90 min at 38 °C before a 1-h recovery at 25 °C. Control larvae were maintained at 25 °C.
Larvae were quick-frozen in liquid nitrogen and lysates were prepared with RIPA buffer supplemented
with 1 mM sodium orthovanadate and 25 mM sodium fluoride. Lysates were cleared by centrifugation
at 14,000g for 15 min and incubated for 4 h at 4 °C with protein G–agarose beads (Roche) pre-bound
to mouse anti-gigas antibodies. Immunoprecipitates were washed five times in RIPA buffer and
resolved on 6% SDS–PAGE gels. Western blots were performed using rabbit anti-phospho-serine
(1:500; Zymed, San Francisco, CA) and rabbit anti-phospho-threonine (1:500; Zymed) antibodies.
Blots were stripped and reblotted with mouse anti-gigas (1:20) antibodies.
Akt in vitro kinase assayThe Akt kinase assay was performed in accordance with the manufacturer’s instructions (Upstate
Biotechnology, Waltham, MA), using 20 mU active recombinant human Akt per reaction and 2.5 µg
GST–Tsc1 or GST–Tsc2 fusion proteins bound to glutathione–Sepharose beads as substrate. After
10 min at 30 °C, the beads were washed once with assay buffer, resolved on a 6% SDS–PAGE gel and
visualized by autoradiography. Recombinant Akt was inactivated by a 10-min incubation at 70 °C.
Data analysisTo calculate ommatidia number, scanning electron micrographs (SEMs) from at least three eyes from
three different UAS-Tsc2 transgenic insertions were analysed for each genotype. To calculate omma-
tidia size, the area covered by a six-by-six ommatidial region was calculated using the Measure func-
tion of NIH Image version 1.62. SEMs from at least four eyes from three independent UAS-Tsc2 trans-
genic insertions were analysed for each genotype. To calculate bristle size, the area covered by the bris-
tle above the socket was measured using NIH Image. Bristle area was compared for both overexpres-
sion clones and immediate wild-type neighbouring bristles. At least five wild-type and overexpression
bristles from at least five wings were analysed for each genotype. Statistical analyses were performed
using Microsoft Excel 98.
RECEIVED 21 MARCH 2002; REVISED 14 JUNE 2002; ACCEPTED 11 JULY 2002;
PUBLISHED 12 AUGUST 2002.
1. Gomez, M. R., Sampson, J. R. & Whittemore, V. H. Tuberous Sclerosis Complex (Oxford University
Press, New York, 1999).
2. Gomez, M. R. Tuberous Sclerosis (Raven Press, New York, 1988).
3. van Slegtenhorst, M. et al. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.
Science 277, 805–808 (1997).
4. Consortium, T. E. C. T. S. Identification and characterization of the tuberous sclerosis gene on
chromosome 16. Cell 75, 1305–1315 (1993).
5. Maheshwar, M. M. et al. The GAP-related domain of tuberin, the product of the TSC2 gene, is a
target for missense mutations in tuberous sclerosis. Hum. Mol. Genet. 6, 1991–1996 (1997).
6. Cheadle, J. P., Reeve, M. P., Sampson, J. R. & Kwiatkowski, D. J. Molecular genetic advances in
tuberous sclerosis. Hum. Genet. 107, 97–114 (2000).
7. Lupas, A., Van Dyke, M. & Stock, J. Predicting coiled coils from protein sequences. Science 252,
1162–1164 (1991).
8. van Slegtenhorst, M. et al. Interaction between hamartin and tuberin, the TSC1 and TSC2 gene
products. Hum. Mol. Genet. 7, 1053–1057 (1998).
9. Plank, T. L., Yeung, R. S. & Henske, E. P. Hamartin, the product of the tuberous sclerosis 1 (TSC1)
gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles. Cancer Res. 58,
4766–4770 (1998).
10. Nellist, M. et al. Characterization of the cytosolic tuberin–hamartin complex. Tuberin is a cytosolic
chaperone for hamartin. J. Biol. Chem. 274, 35647–35652 (1999).
11. Miloloza, A. et al. The TSC1 gene product, hamartin, negatively regulates cell proliferation.
Hum. Mol. Genet. 9, 1721–1727 (2000).
12. Xu, T., Wang, W., Zhang, S., Stewart, R. A. & Yu, W. Identifying tumor suppressors in genetic
mosaics: the Drosophila lats gene encodes a putative protein kinase. Development 121, 1053–1063
(1995).
13. Theodosiou, N. A., Zhang, S., Wang, W. Y. & Xu, T. slimb coordinates wg and dpp expression in the
dorsal–ventral and anterior–posterior axes during limb development. Development 125, 3411–3416
(1998).
14. Ito, N. & Rubin, G. M. gigas, a Drosophila homolog of tuberous sclerosis gene product-2, regulates
the cell cycle. Cell 96, 529–539 (1999).
15. Huang, H. et al. PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye devel-
opment. Development 126, 5365–5372 (1999).
16. Oldham, S., Montagne, J., Radimerski, T., Thomas, G. & Hafen, E. Genetic and biochemical charac-
terization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14, 2689–2694
(2000).
17. Gao, X., Neufeld, T. P. & Pan, D. Drosophila PTEN regulates cell growth and proliferation through
PI3K-dependent and -independent pathways. Dev. Biol. 221, 404–418 (2000).
18. Potter, C. J., Huang, H. & Xu, T. Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling
in regulating cell growth, cell proliferation, and organ size. Cell 105, 357–368 (2001).
19. Tapon, N., Ito, N., Dickson, B. J., Treisman, J. E. & Hariharan, I. K. The Drosophila tuberous sclero-
sis complex gene homologs restrict cell growth and cell proliferation. Cell 105, 345–355 (2001).
20. Gao, X. & Pan, D. TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth.
Genes Dev. 15, 1383–1392 (2001).
21. Moberg, K. H., Bell, D. W., Wahrer, D. C., Haber, D. A. & Hariharan, I. K. Archipelago regulates
Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature 413, 311–316
(2001).
22. Goberdhan, D. C., Paricio, N., Goodman, E. C., Mlodzik, M. & Wilson, C. Drosophila tumor sup-
pressor PTEN controls cell size and number by antagonizing the Chico/PI3-kinase signaling path-
way. Genes Dev. 13, 3244–3258 (1999).
23. Maehama, T. & Dixon, J. E. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid sec-
ond messenger, phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273, 13375–13378 (1998).
24. Cantley, L. C. & Neel, B. G. New insights into tumor suppression: PTEN suppresses tumor forma-
tion by restraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl Acad. Sci. USA 96,
4240–4245 (1999).
25. Lehner, C. F. The beauty of small flies. Nature Cell Biol. 1, E129–E130 (1999).
26. Edgar, B. A. From small flies come big discoveries about size control. Nature Cell Biol. 1, E191–E193
(1999).
27. Weinkove, D. & Leevers, S. J. The genetic control of organ growth: insights from Drosophila.
Curr. Opin. Genet. Dev. 10, 75–80 (2000).
28. Stocker, H. & Hafen, E. Genetic control of cell size. Curr. Opin. Genet. Dev. 10, 529–535 (2000).
29. Alessi, D. R. & Cohen, P. Mechanism of activation and function of protein kinase B. Curr. Opin.
Genet. Dev. 8, 55–62 (1998).
30. Brazil, D. P. & Hemmings, B. A. Ten years of protein kinase B signalling: a hard Akt to follow.
Trends Biochem. Sci. 26, 657–664 (2001).
31. Scheid, M. P. & Woodgett, J. R. PKB/AKT: functional insights from genetic models. Nature Rev.
Mol. Cell Biol. 2, 760–768 (2001).
32. Verdu, J., Buratovich, M. A., Wilder, E. L. & Birnbaum, M. J. Cell-autonomous regulation of cell
and organ growth in Drosophila by Akt/PKB. Nature Cell Biol. 1, 500–506 (1999).
33. Shioi, T. et al. Akt/protein kinase B promotes organ growth in transgenic mice. Mol. Cell. Biol. 22,
2799–2809 (2002).
34. Stambolic, V. et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppres-
sor PTEN. Cell 95, 29–39 (1998).
35. Aoki, M., Blazek, E. & Vogt, P. K. A role of the kinase mTOR in cellular transformation induced by
the oncoproteins P3k and Akt. Proc. Natl Acad. Sci. USA 98, 136–141 (2001).
36. Alessi, D. R., Caudwell, F. B., Andjelkovic, M., Hemmings, B. A. & Cohen, P. Molecular basis for the
substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase.
FEBS Lett. 399, 333–338 (1996).
37. Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generat-
ing dominant phenotypes. Development 118, 401–415 (1993).
38. Scanga, S. E. et al. The conserved PI3′K/PTEN/Akt signaling pathway regulates both cell size and
survival in Drosophila. Oncogene 19, 3971–3977 (2000).
© 2003 Nature Publishing Group
articles
NATURE CELL BIOLOGY VOL 4 SEPTEMBER 2002 http://www.nature.com/naturecellbiology 665
39. Ito, K., Awano, W., Suzuki, K., Hiromi, Y. & Yamamoto, D. The Drosophila mushroom body is a
quadruple structure of clonal units each of which contains a virtually identical set of neurones and
glial cells. Development 124, 761–771 (1997).
40. Struhl, G. & Basler, K. Organizing activity of wingless protein in Drosophila. Cell 72, 527–540 (1993).
41. Schmelzle, T. & Hall, M. N. TOR, a central controller of cell growth. Cell 103, 253–262 (2000).
42. Dufner, A. & Thomas, G. Ribosomal S6 kinase signaling and the control of translation. Exp. Cell
Res. 253, 100–109 (1999).
43. Bunch, T. A., Grinblat, Y. & Goldstein, L. S. Characterization and use of the Drosophila metalloth-
ionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res. 16, 1043–1061 (1988).
44. Halder, G. et al. Eyeless initiates the expression of both sine oculis and eyes absent during Drosophila
compound eye development. Development 125, 2181–2191 (1998).
45. Hay, B. A., Maile, R. & Rubin, G. M. P element insertion-dependent gene activation in the
Drosophila eye. Proc. Natl Acad. Sci. USA 94, 5195–5200 (1997).
46. Zhang, H., Stallock, J. P., Ng, J. C., Reinhard, C. & Neufeld, T. P. Regulation of cellular growth by
the Drosophila target of rapamycin dTOR. Genes Dev. 14, 2712–2724 (2000).
47. Fehon, R. G. et al. Molecular interactions between the protein products of the neurogenic loci
Notch and Delta, two EGF-homologous genes in Drosophila. Cell 61, 523–534 (1990).
ACKNOWLEDGEMENTS
We thank K. Wehner for helpful discussions, M. Birnbaum for Dakt1 reagents, the Developmental
Studies Hybridoma bank for anti-gigas antibodies and X. Fei for injections. This work was supported
in part by a National Institutes of Health grant (CA69408) and the Rothberg Courage Award, TS
Alliance. T.X. is an investigator of the Howard Hughes Medical Institute. C.J.P. was a predoctoral can-
didate in the Department of Genetics.
Correspondence and requests for materials should be addressed to T.X.
Supplementary Information accompanies the paper on www.nature.com/naturecellbiology.
COMPETING FINANCIAL INTERESTS
The authors declare that they have no competing financial interests.
© 2003 Nature Publishing Group
supplementary infomation
1
Figure S1 Akt expression does not enhance the Tsc1 mutant phenotype. a, Aktexpression leads to an increase in ommatidia size. b, A mosaic eye containing Tsc1mutant ommatidia is shown. Mutation of Tsc1 also leads to an increase in ommatidiasize18,19. c, Ectopic expression of Akt does not increase the size of the Tsc1 mutantommatidia. Genotypes: a, yw, ey-FLP; pGMR-GAL4/UAS-Dakt1; FRT82B P[mini-w+]88C/TM6B; b, yw, ey-FLP; pGMR-GAL4/CyO; FRT82B Tsc1Q600X/FRT82B P[mini-
w+]88C; c, yw, ey-FLP; pGMR-GAL4/UAS-Dakt1; FRT82B Tsc1Q600X/FRT82B P[mini-w+]88C. Higher magnifications of the boxed regions are in the lower panel. Tsc1mutant clones are outlined in white. Inset values represent the mean ommatidiasize ± standard deviation in arbitrary units. At least 30 ommatidia of each genotypewere measured.
© 2003 Nature Publishing Group
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