phosphatidylinositol3-kinase-dependentactivationof ...may 15, 2009•volume 284•number 20 journal...

Phosphatidylinositol 3-Kinase-dependent Activation ofMammalian Protein Kinase B/Akt in Saccharomyces cerevisiae,an in Vivo Model for the Functional Study of Akt Mutations*

Received for publication, October 14, 2008, and in revised form, March 17, 2009 Published, JBC Papers in Press, March 23, 2009, DOI 10.1074/jbc.M807867200

Isabel Rodríguez-Escudero‡, Amparo Andres-Pons§1, Rafael Pulido§, María Molina‡, and Víctor J. Cid‡2

From the ‡Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040 and the§Centro de Investigacion Príncipe Felipe, Valencia 46012, Spain

In animal cells, Akt (also called protein kinase B) is activatedby stimuli that elevate the level of phosphatidylinositol 3,4,5-trisphosphate and is amajor effector for eliciting responses thatsupport cell growth and survival.Wehave shownpreviously thatco-expression of Akt1 in budding yeast (Saccharomyces cerevi-siae) along with hyperactive p110�, the catalytic subunit ofmammalian phosphatidylinositol 3-kinase, results in Akt1 re-localization to cellular membranes and activation. In the pres-ent study, we show that activation of all three mammalian Aktisoforms by wild-type p110� causes deleterious effects on yeastcell growth. Toxicity of Akt in S. cerevisiae required its catalyticactivity, its pleckstrin homology domain, and phosphorylationof its activation loop, but not phosphorylation of its hydropho-bic motif. We demonstrate that expression in yeast of the onlypurported oncogenic allele, Akt1(E17K), leads to enhanced phe-notypes. Ala-scanningmutagenesis of theVL1 regionwithin thephosphatidylinositol 3,4,5-trisphosphate-interacting pocket ofthe Akt1 pleckstrin homology domain revealed that most resi-dues in this region are essential for Akt1 activity.We found thatactive Akt leads to enhanced signaling through the yeast cellwall integrity pathway. This effect requires the upstream Rho1activatorRom2and involves bothphosphorylationof theMAPKSlt2 and expression of its transcriptional targets, thus providinga quantitative reporter system for heterologous Akt activity invivo. Collectively, our results disclose a heterologous yeast sys-tem that allows the functional assessment in vivo of both loss-of-function and tumorigenic Akt alleles.

Protein kinase B (PKB,3 also known as cellular Akt or c-Akt)belongs to the AGC (cAMP-dependent, cGMP-dependent,

protein kinase C) family of protein kinases and is considered acentral player in regulation of metabolism, cell survival, apo-ptosis, cellular proliferation, motility, transcription and cell-cycle progression (1, 2). The Akt subfamily comprises threemammalian isoforms, Akt1, Akt2, and Akt3 (PKB�, PKB�, andPKB�, respectively), whereas invertebrates, like flies andworms, have a single PKB/Akt protein. The relative expressionof the three mammalian isoforms of Akt differs in differenttissues: Akt1 is the predominant isoform in the majority of tis-sues, Akt2 is mainly present in insulin-responsive tissues, andAkt3 prevails in brain and testes. The three mammalian Aktmembers are products of different genes and share a commonstructure that consists of three conserved domains: an N-ter-minal pleckstrin homology (PH) domain, a kinase catalyticdomain at the center, and a C-terminal regulatory domain con-taining the hydrophobic motif (HM) phosphorylation site(FXXF(S/T)Y) (3, 4). In the lower eukaryote Saccharomyces cer-evisiae, an AGC protein kinase, Sch9, has been proposed as afunctional ortholog ofmammalianAkt, and is involved in nutri-ent sensing, ribosome biogenesis, lifespan, adaptation toosmotic stress, and cell-size control (5–10). In higher cells, Aktmembers transduce signals downstream the class I phospha-tidylinositol 3-kinases (PI3Ks), which are also proto-oncogenicproteins. Class I PI3K are heterodimeric enzymes containing acatalytic subunit (p110�, -�, -�, or -�) and a regulatory subunit(p85�, p85�, p55�, or p101). Activation of p110 catalytic sub-units takes place by growth factor- and hormone-binding totyrosine kinase- and G protein-coupled receptors, and involvesthe recruitment of the PI3K regulatory subunits to Tyr-phos-phorylated receptors and scaffolding signaling proteins, as wellas binding to Ras. The activated PI3K converts membranebound phosphatidylinositol 4,5-bisphosphate (PIP2) to phos-phatidylinositol 3,4,5-trisphosphate (PIP3). The structure ofthe PH domain of Akt conforms a pocket that accommodatesthe polar head group of PIP3 (11). This interaction results in thetranslocation of the Akt kinase from the cytoplasm to theplasma membrane, where it undergoes activation. Mutationalanalyses have suggested that membrane localization is a pri-mary requirement for Akt oncogenicity andmediation of PI3K-dependent signaling (12, 13). Akt activity is negatively regulatedby phosphatidylinositol phosphatases that dephosphorylatePIP3. The major PI3K down-regulator is the PTEN phospha-

* This work was supported in part by Grant BIO2007-67299 from MinisterioEducacion y Ciencia, Grant S-SAL-0246-2006 and the Programme for Uni-versidad Complutense de Madrid Research Groups (920628) from Comu-nidad Autonoma de Madrid (to M. M.) and by Grant SAF2006-083139 fromMinisterio de Educacion y Ciencia, Grant ISCIII-RETIC RD06/0020 from Insti-tuto de Salud Carlos III, Spain, and Fundacion Mutua Madrilena (to R. P.).

1 Recipient of a predoctoral fellowship from Ayuntamiento de Valencia,Spain.

2 To whom correspondence should be addressed: Departamento de Microbi-ología II, Facultad de Farmacia, Universidad Complutense de Madrid, Pza.Ramon y Cajal s/n 28040, Madrid, Spain. Tel.: 34-91-394-1888; Fax: 34-91-394-1745; E-mail: [email protected].

3 The abbreviations used are: PKB, protein kinase B; PH, pleckstrin homology;HM, hydrophobic motif; PI3K, phosphatidylinositol 3-kinase; PIP2, phos-phatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PTEN, phosphatase and tensin homolog; PDK1, phospho-inositide-dependent kinase 1; mTOR, mammalian target of rapamycin;

GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase;MAPKKK, MAPK kinase kinase; wt, wild type; CWI, cell wall integrity.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 20, pp. 13373–13383, May 15, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

MAY 15, 2009 • VOLUME 284 • NUMBER 20 JOURNAL OF BIOLOGICAL CHEMISTRY 13373

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tase, which is considered a tumor suppressor because loss-of-function mutations are found in overt tumors (14) as well as inthe germ line in hereditary diseases that predispose to cancer(15). PTEN dephosphorylates the 3�-phosphate of PIP3 andthereby negates the activity of PI3K (16). PIP3 is also metabo-lized to phosphatidylinositol 3,4-bisphosphate by 5-phospho-inositide phosphatases, like SHIP1 and SHIP2 (SH2-containinginositol phosphatase-1 and -2); but PIP3 and phosphatidyl ino-sitol 3,4 bisphosphate mostly engage the same effectors. Oncerecruited to the plasma membrane, Akt is phosphorylated attwo critical residues for its full activation. These residuesinclude a Thr residue in the activation loop within the kinasecatalytic domain (Thr-308, Thr-309, and Thr-305 for Akt1,Akt2, and Akt3, respectively) and a Ser residue on the hydro-phobic motif (HM; Ser-473, Ser-474, and Ser-472 for Akt1,Akt2, and Akt3, respectively). The activation loop kinase forAkt is the phosphoinositide-dependent kinase 1 (PDK1). TheHM kinase, also termed PDK2, has been identified recently asmTORC2 (17). The full activation of Akt permits a conforma-tional change that results in substrate binding and a significantincrease of the rate of catalysis.The relevance of this pathway in clinical oncology has been

evidenced by the frequency of mutations in its major down-regulator, the tumor suppressor PTEN (18) and, more recently,by the isolation of oncogenic mutations in both PI3K and Akt1themselves (19–21). In previous reports, we described that het-erologous expression of PI3K, PTEN, and Akt1 in S. cerevisiaereproduces several aspects of their function, including PIP3-de-pendent membrane translocation and phosphorylation of Akt1at both activation residues by the yeast PDK1 orthologs and anunidentified endogenous kinase (22). We have also reportedpreviously that this heterologousmodel system could be readilyexploited to evaluate oncogenicity of both PI3K and PTENmutants (23). Here, we show that catalytically active mamma-lian Akt inhibits yeast cell growth, thus extending the system toperform structure-function relationship studies on Akt in vivo.

EXPERIMENTAL PROCEDURES

Strains, Media, and Growth Conditions—The S. cerevisiaestrains used in the present study were YPH499 (MATa ade2–101 trp1–63 leu2–1 ura3–52 his3–200 lys2–801, P. Hieter),BY4741 (MATa his3�1; leu2�; met15�; ura3�, EUROSCARF,Germany), slt2� (BY4741 isogenic, slt2::KanMX4) bck1�(BY4741 isogenic, bck1::KanMX4), and rom2� (BY4741 iso-genic, rom2::KanMX4). Escherichia coli DH5� F�[K12� (lac-ZYA-argF)U169 deoR supE44 thi-1 recA1 endA1 hsdR17gyrA96 relA1 (� 80lacZ � M15)F�] was used for routine molec-ular biology techniques. YPD (1% (w/v) yeast extract, 2% (w/v)peptone, and 2% (w/v) dextrose/glucose) broth or agar was thegeneral non-selective medium used for yeast cell growth. Syn-thetic minimal medium (SD) contained 0.17% yeast nitrogenbase without amino acids, 0.5% ammonium sulfate, and 2% glu-cose and lacked appropriate amino acids and nucleic acid basesto maintain selection for plasmids. In SG (synthetic galactose)and SR (synthetic raffinose) media, glucose was replaced with2% (w/v) galactose or 2% (w/v) raffinose, respectively. Galactoseinduction in liquid medium was performed by growing cells inSR to mid-exponential phase and then adding galactose to 2%

for 4–6 h. Growth assays on plates were performed, typically,by growing transformants overnight in SD lacking leucine anduracil, or leucine and tryptophan, adjusting the culture to anabsorbance A600 0.5, and spotting samples (5 �l) of the cellsuspension and three serial 10-fold dilutions on to the surface ofSD or SG plates lacking the appropriate nutrients to maintainselection for plasmids, followed by incubation at 30 °C for 2–3days. The mTOR inhibitor rapamycin (Sigma-Aldrich) wasadded to SG plates lacking the appropriate nutrients at a finalconcentration of 0.2 �g/ml.Plasmid Construction—Transformation of E. coli and

yeast and other basic molecular biology methods were car-ried out using standard procedures. Plasmids pYES2-PTEN,pYES2-GFP-Akt1, pYES-GFP-AktK179M, pYES2-myr-Akt1,YCpLG-myc-p110�-CAAX, YCpLG-myc-p110�-wt, YCpLG-myc-p110�-E545K, YCpLG-myc-p110�-H1047R, and pYES3-GFP-Akt1 have been described (22, 23). pYES2-SHIP1was con-structed by subcloning human SHIP1 into pYES2 by PCR frompOTB7 SHIP1 (Mammalian Gene Collection, IMAGE ID6304992, Geneservice, UK). To produce pYES2-GFP-Akt2, ratAkt2 coding sequence was amplified by PCR from the pEGFP-Akt2 plasmid (24) with primers bearing the appropriatedrestriction sites at their 5� tails and cloned in-frame into theEcoRI/XbaI sites of pYES2-GFP. The latter plasmid had beenconstructed by amplifying the GFP sequence by PCR and clon-ing it into the HindIII/BamHI sites of pYES2. For pYES2-GFP-Akt3, the mouse Akt3 coding sequence was amplified by PCRfrom pSPORT1 Akt3 (Mammalian Gene Collection, IMAGEID 30089997, Geneservice, UK) as above and cloned EcoRI-XbaI into pYES2-GFP. The primers used for amplification hadEcoRI- or XbaI-containing tails in the upper and in the loweroligonucleotides, respectively, to allow directed cloning intothe same sites of the pYES2-GFP. YCpLG-myc-p110� andYCpLG-myc-p110� were made by PCR from the plasmidspCMVSPORT6 p110� (human sequence, Mammalian GeneCollection, IMAGE ID 5749986) and pCMVSPORT6 p110�(mouse sequence, Mammalian Gene Collection, IMAGE ID4192906), respectively. To produce plasmids pYES3-GFP-Akt1R25A, pYES3-GFP-Akt1E40K, pYES3-GFP-Akt1T308A, andpYES3-GFP-Akt1S473A site-directed mutagenesis was carriedout using standard full plasmid amplification by DpnI-basedPCR strategy with Turbo PfuI DNA polymerase (Stratagene)using pYES3-GFP-Akt1 as template. To obtain the plasmidpYES3-GFP-Akt1T308A,S473A we performed the same strat-egy using the pYES3-GFP-Akt1S473A plasmid as template.To obtain the plasmids pYES2-GFP-Akt1E17K, pYES2-GFP-Akt1T308D, pYES2-GFP-Akt1S473D, pYES2-GFP-Akt1F469A,F472A, pYES2-GFP-Akt1Y474A, pYES2-GFP-Akt11–467, and pYES2-GFP-Akt11–454 the same strategy wasperformed, but using pYES2-GFP-Akt1 as template. Toobtain the plasmid pYES2-GFP-Akt1T308D,S473D we per-formed the same strategy using the pYES2-GFP-Akt1S473Dplasmid as template. The Ala scanning collection of mutants(R15A, G16A, E17A, Y18A, I19A, K20A, T21A, W22A,R23A, and P24A), as well as the G16R and P24R mutations,were also obtained by PCR-based site-directed mutagenesison pYES2-GFP-Akt1. The pYES2-GFP-Sch9 plasmid wasconstructed by PCR amplification from the Sch9 open read-

Activation of Mammalian Akt in Yeast

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ing frame from S. cerevisiae BY4741 genomic DNA. Theprimers used for amplification had BamHI- or EcoRI-con-taining tails in the upper and in the lower oligonucleotides,respectively, to allow directed cloning. PCR products werecleaved with BamHI-EcoRI to be inserted in the same sites ofthe pYES2-GFP vector. To generate the chimeras pYES2-Sch9-Akt1 and pYES2-Akt1-Sch9, standard sequential PCR-based method (25) was performed. The primers used foramplification of the different Sch9 or Akt1 regions hadBamHI- or EcoRI-containing tails in the upper and loweroligonucleotides, respectively, to allow directed cloning.PCR products were cleaved with BamHI/EcoRI to beinserted in the same sites of the pYES2-GFP plasmid. ThepMLP1-LACZ (URA3) plasmid (26) and its HIS3-based ver-sion were a gift of A. B. Sanz, R. García, and J. Arroyo. Oli-gonucleotide sequences used in this work are available uponrequest. In all cases, fidelity of the amplified DNA was veri-fied by DNA sequencing.Immunodetection by Western Blotting—The standard proce-

dures were used for yeast cell growth, collection, and breakage,collection of proteins, fractionation by SDS-polyacrylamide gelelectrophoresis, and transfer to nitrocellulosemembranes. Rabbitanti-phospho-Akt1 (P-Thr-308) and rabbit anti-phospho-Akt1(P-Ser-473) (Cell Signaling Technology) were used to detect thephosphorylation of the corresponding sites in PKB/Akt or the chi-merical protein Sch9-Akt1. GFP fusion proteins were detectedusing amouse anti-GFP antibody (JL-8, BD Biosciences, San Jose,CA).Yeast actinwasdetectedby cross-reactionwith amouse anti-actin monoclonal antibody (Clone C4, MP Biomedicals, Irvine,CA).Afterwashing,boundprimaryantibodieswererevealedusinghorseradish peroxidase-conjugated anti-rabbit or anti-mouse sec-ondary antibodies, as appropriate, and a chemiluminescencedetection system (ECL, Amersham Biosciences).Microscopy Techniques—For fluorescence microscopy on

live cells (for the observation of GFP), cells from exponentiallygrowing cultures were harvested by brief centrifugation,washed once with phosphate-buffered saline, and vieweddirectly. To monitor PI3K-dependent membrane localizationof GFP-Akt, a time course was made to monitor membranebinding after induction of both p110 and GFP-Akt in galactose.Localization to membranes was significant at t � 4 h (�60%),and reached the maximum at t � 8 h (�90%). To allow detec-tion of both positive and negative changes in GFP-Akt affinityfor membranes, all data presented here were analyzed at 4 hafter galactose induction. For statistics on cell populations,an average of �200 cells were counted for each experiment.Cells were examined with an Eclipse TE2000U microscope(Nikon) using the appropriate sets of filters. Digital imageswere acquired with Orca C4742-95-12ER charge-coupleddevice camera (Hamamatsu) and Aquacosmos Imaging Sys-tems software.

�-Galactosidase Assays—Yeast cell extracts were preparedby harvesting cells by centrifugation from 10ml of an exponen-tially growing culture after induction with galactose for 6 h.Then, cells were resuspended in 250 �l of breaking buffer (100mM Tris-HCl, pH 8, 1 mM dithiothreitol, 20% glycerol), andglass beads (Glasperlen,�1mm, Sartorius AG, Germany) wereadded to break cells in a Fast-Prep machine. Finally, extracts

were clarified by centrifugation, and protein concentrationswere measured using the Bradford method. �-Galactosidaseassays were performed using the crude extracts obtained asdescribed previously (27), scaling the protocol to a 96-wellmicrotiter plate format. 10 �l of cell extract was mixed with 90�l of Z buffer plus �-mercaptoethanol (0.03%) and 20 �l ofo-nitrophenyl-�-D-galactopyranoside (4 mg/ml in Z buffer).The absorbance of the enzymatic reaction was measured at415 nm on a microplate reader (Model 680, Bio-Rad) after atleast 10 min of incubation at 30 °C and after the addition of50 �l of 1 M Na2CO3 to stop the reaction. �-Galactosidaseactivity was expressed as nanomoles of o-nitrophenyl-�-D-galactopyranoside converted/min/mg of protein. Experi-ments were performed at least three times from independentyeast transformants.

RESULTS

In Vivo Activated Mammalian Akt1 Impairs Yeast Growth—In previous reports, we have shown that expression of mem-brane-targeted mammalian class I PI3K catalytic subunit(p110�-CAAX) driven by the galactose-inducible GAL1 pro-moter inhibited yeast growth, mainly by depletion of essentialphosphatidylinositol-4,5-bisphosphate (PIP2) pools (22). How-ever,WT p110� in the same expression system had no negativeeffects on growth (23) (Fig. 1A). Remarkably, co-expression of

FIGURE 1. Akt1 inhibits growth of yeast cells when activated in vivo.A, growth of the yeast WT strain (YPH499) is impaired by expression of Akt1 inthe presence of WT or tumor-related E545K and H1047R mutations of thecatalytic subunit of PI3K, p110�. GFP-Akt1 was expressed from pYES2-GFP-Akt1 URA3-based plasmid under the control of the galactose-inducible GAL1promoter, and all versions of p110� were expressed from LEU2-marked vec-tors of the YCpLG-myc-p110� series under the control of the same promoter.Serial 10-fold dilutions of cultures of representative transformants were spot-ted on synthetic medium lacking uracil and leucine under repressing (glu-cose; SD medium) or inducing (galactose; SG medium) conditions, as indi-cated. �, designates the corresponding YCpLG or pYES2-GFP empty vectors.B, Akt1-induced growth inhibition is counterbalanced by the 3-phosphatidyl-inositol phosphatase PTEN, but not by the 5-phosphatidylinositol phospha-tase SHIP1. Akt1 was expressed from the TRP1-based pYES3-GFP-Akt1 plas-mid, p110� from the YCpLG-myc-p110� plasmid, and PTEN or SHIP1 frompYES2-PTEN and pYES2-SHIP1, respectively, all under the control of the GAL1promoter. Serial 10-fold dilutions were spotted as above on selective medialacking uracil, tryptophan, and leucine. “Vector” indicates the pYES3 emptyplasmid.




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WT p110� with GFP-Akt1 led to poor growth, an effect thatwas more intense with the oncogenic p110� H1047R mutantprotein, described to have an enhanced kinase activity (19, 28)(Fig. 1A). In contrast, the oncogenic mutation E545K, whichowes its effect to abnormal interactionwith the PI3K regulatorysubunit p85 rather than to enhanced lipid kinase activity (29,30), behaved like WT p110� regarding growth impairment inGFP-Akt1-expressing yeast cells (Fig. 1A). Thus, dose-depend-ent PIP3-driven Akt activation can be reproduced in yeast, and,furthermore, it could bemonitored by analyzing its toxic effect.If it is true that Akt1 toxicity depends on intracellular PIP3levels, it should be relieved by co-expression of the tumor sup-pressor PTEN, which operates as a PIP3 3-phosphatase. Indeed,as shown in Fig. 1B, co-expression of PTEN in cells expressingp110� andAkt1 rescued growth, while co-expression of SHIP1,a PIP3 5-phosphatase that generates phosphatidylinositol 3,4-bisphosphate instead of PIP2, was unable to revert the p110�-induced Akt1 toxicity. This result is in agreement with in vitroand structural data (11, 31) that support the idea that Akt1 isable to respond similarly to both PIP3 and phosphatidylinositol3,4-bisphosphate.Toxicity ofAkt1 inYeastRequires ItsKinaseActivity andPIP3-

dependentMembrane Recruitment—Weused aK179Mkinase-dead version of Akt1 to understand whether toxicity in yeastwas due to its protein kinase activity. As shown in Fig. 2A, co-expression of p110� and Akt1K179M had no effect on yeastgrowth, bringing forward the conclusion that impairment ofgrowth is a consequence of the phosphorylation of endogenousyeast protein targets by Akt1. Also, we expressed amyristoylat-able version of Akt1, which is directed to membranes inde-pendently of PI3K-generated PIP3 (22). Peculiarly, myr-GFP-

Akt1, extensively used as a constitutively active kinase inmammalian cells, was neither toxic in the absence nor in thepresence of p110� (Fig. 2A), indicating that it cannot be used asa constitutively active Akt version in ourmodel. This is consist-ent with our previous observation that myr-GFP-Akt1 is lessefficiently phosphorylated than GFP-Akt1 in the yeast cell (22)and suggests that myr-GFP-Akt may be less available as a sub-strate for its activating endogenous kinases. Thus, the concen-tration of activated Akt1 at the particular spots where PIP3 isgenerated from endogenous PIP2 pools, rather than its indis-criminate attachment to membranes, seems a requirement forits toxicity in the yeast cell.In mammalian cells, key effects of PI3K-dependent Akt acti-

vation related to control of cellular proliferation and survivalrely on their downstream effector, the mammalian target ofrapamycin (mTOR). Yeast Tor1 seems to have a significantdegree of functional conservation with respect to its mamma-lian counterpart (32). However, inhibition of growth inducedby PI3K and Akt1 in yeast was unaffected by the presence ofrapamycin (Fig. 2B). Although we cannot discard that heterol-ogous Akt might couple to Tor signaling in yeast, this resultindicates that Akt1-induced toxicity is not mediated by theyeast rapamycin-dependent TORC1 complex.All Akt Isoforms Respond to PIP3 Production in Yeast Impair-

ing Cell Growth—Next we tested in the yeast system other iso-forms of PKB/Akt besides PKB�/Akt1, namely PKB�/Akt2 andPKB�/Akt3, by generating the corresponding fusions to GFP inthe same expression vectors. All three Akt isoforms behavedequivalently in terms of p110�-dependent growth inhibition(Fig. 3A) and PIP3-dependent localization to the plasma mem-brane (data not shown). Upon p110� co-expression, Akt2 andAkt3 displayed enhanced phosphorylation at the activationsites equivalent to Thr-308 and Ser-473 in Akt1 (Fig. 3B). Thisindicates that toxicity of all Akt isoforms in yeast correlates totheir PIP3-dependent activation in vivo.We also tested the fourisoforms ofWT p110 (�, �, �, and �). However, only p110� wascapable of inducing toxicity when co-expressed with any Aktisoform (data not shown), suggesting that p110� is a morerobust enzyme in vivo than the other isoforms.Phosphorylation of Akt1 at Thr-308, but Not Ser-473, Is

Essential for Toxicity in Yeast—Toattest the contribution of thephosphorylation of Thr-308 and Ser-473 to activation of Akt inthe yeast model, we mutated both residues to Ala in Akt1 bysite-directedmutagenesis.Thesemutationsdidnot affectPI3K-dependent re-localization of GFP-Akt1 to the yeast plasmamembrane (data not shown). As expected,mutation of Thr-308to Ala greatly eliminated toxicity of GFP-Akt1 (Fig. 4A), indi-cating that phosphorylation of this residue by yeast PDK1orthologs is crucial for the activation of the Akt1 kinase in vivoin the yeast model. However, unexpectedly, the S473A muta-tion did not affect Akt1 toxicity, and a double T308A/S473Amutant behaved like the single T308A mutant (Fig. 4A). Thisimplies that the observed phosphorylation of Akt1 at the HM isnot linked to its effects on the yeast cell. Analyses of Akt1P-Thr-308 and P-Ser-473 content in the T308A and S473Amutants verified that the corresponding epitopes were missingand revealed that phosphorylation of any of these two residuesin yeast in the absence of the other was lower than inWTAkt1,

FIGURE 2. Akt1 effects in yeast require an active kinase and are independ-ent of rapamycin. A, mutation of the catalytic residue Lys-179 in Akt1 or itsconstitutive targeting to cellular membranes eliminates toxicity. GFP-Akt1,GFP-Akt1K179M, and myr-GFP-Akt1 were expressed from the correspondingpYES2-Akt1 vectors, and p110� from YCpLG-myc-p110�. Spotting of cell sus-pensions on agar media was performed as in Fig. 1A. B, Akt1 effects are notprecluded by the presence of rapamycin. Akt1 and Akt1K179M were expressedfrom the corresponding pYES2-GFP-Akt1 plasmids, and p110� from YCpLG-myc-p110�. Serial 10-fold dilutions were spotted as above, except that the SGplates also contained rapamycin at a final concentration of 0.2 �g/ml, asindicated.




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but still responsive to p110� (Fig. 4B). We also generated andtestedmutants that had each or both Thr-308 and Ser-473 sitesin Akt1 mutated to Asp, a putative phospho-mimetic residue.We observed that the behavior of GFP-Akt1T308D and GFP-Akt1S473D was very similar to that of the mutants to Ala (datanot shown), that is, the T308D mutation eliminated toxicity,whereas the S473Dmutation did not add any effect. This mightreflect that recognition by the endogenous PDK1-like kinasesin situ is related to toxicity. Nevertheless, as shown in Fig. 4C,neither the T308D mutation interfered with Ser-473 phospho-rylation, nor S473D had any effect on Thr-308 phosphoryla-tion, whereas the equivalent changes to Ala negatively influ-enced each other phosphorylation. This might suggest thatmutations to Asp lead to amore favorable conformation for therecognition of Akt1 by its heterologous activating kinases.The observation that phosphorylation of the Ser-473 residue

was dispensable for the toxicity of Akt1 in yeast prompted us tostudy the involvement of the whole C-terminal HM, a regionthat is highly conserved in the three isoforms of Akt (Fig. 4D).The Phe-469, Phe-472, and Tyr-474 residues (Fig. 4D, arrows)that surround Ser-473 have been described to be important forAkt structure and function (33). By site-directed mutagenesis,we generated two mutants with these hydrophobic residuesmutated to Ala: GFP-Akt1F469A,F472A and GFP-Akt1Y474A. Weobserved that toxicity of these mutants in yeast was slightlyreduced as compared with that of WT GFP-Akt1 (Fig. 4E),revealing a limited contribution of the HM to toxicity. We alsogenerated a truncated version of Akt1 bearing a stop codon inposition 468 (GFP-Akt11–467), thus lacking the whole HM.

Moreover, based on the existence of an alternatively splicedform of Akt3, namely Akt3-�1, that lacks a longer stretch at theC terminus of the Akt3 isoform (34) (Fig. 4D), we made a min-imum length truncation of the Akt1 cDNA equivalent to theAkt3-�1 splicing variant (GFP-Akt11–454). Like the HM pointmutants, the truncated versions inhibited yeast growth in ap110�-dependent manner, although slightly less efficiently thanWT GFP-Akt1 (Fig. 4E). For both HM point mutants and trun-cated versions, phosphorylation at the T-loop was still responsiveto PI3K (Fig. 4F). In conclusion, the HM of Akt1 and the 14 resi-dues preceding it, which are missing in a splicing variant of Akt3,contribute only partially to the effects of Akt1 in yeast.The PHDomain of Akt1Confers to the KinaseDomain of Sch9

Responsiveness to PIP3—Suggesting functional conservationbetween AGC kinase-controlled pathways from yeast to mam-mals, Sch9 has been reported to be a putative yeast ortholog ofmammalian Akt (5). Overexpression of Sch9 led to a very mildreduction of yeast growth that was PI3K-independent (Fig. 5A).Actually, suchmild growth inhibition was also observed in cellsoverexpressing Akt1 in the absence of p110�. We studied theinfluence of endogenous PIP3 production in the localization ofGFP-Sch9. Overexpressed GFP-Sch9 was ubiquitous in thecytoplasm, excluded from vacuoles and frequently concen-trated in cytoplasmic spots. Unlike GFP-Akt, re-localization ofGFP-Sch9 to the plasma membrane did not occur when PIP3was produced by p110� (Fig. 5B). Thus, Sch9 was not respon-sive to PIP3. Accordingly, the most striking difference betweenboth AGC kinases is that the N-terminal PIP3-binding PHdomain of Akt is replaced by a sphingosine long chain base-responsive sequence in yeast Sch9 (35, 36). We interchangedthe N-terminal regulatory and C-terminal protein kinasedomains of Sch9 and Akt1 producing the following chimeras:GFP-Akt11–148-Sch9411–824, bearing the Akt1 PH domain andthe Sch9 kinase domain; and GFP-Sch91–410-Akt1149–480,bearing the Sch9 sphingoid-responsive region and the Akt1kinase domain. Expression of the chimeras was equivalent tothat of the wild-type fusions (Fig. 5C). Although the strongp110�-dependent effect of Akt1 on growth did not occur ineither chimerical construct, the Akt1–148-Sch9411–824 had agreater inhibitory effect than Sch9 in the presence of p110�(Fig. 5A). Also, this chimera was efficiently re-located to theplasma membrane when p110� was present (Fig. 5B). In con-sequence, the kinase domain of Sch9 can reproduce the effectsof heterologous Akt1 in yeast, albeit less efficiently. The GFP-Sch91–410-Akt1149–480 chimera behaved like GFP-Sch9 interms of PI3K-independent toxicity and localization. Pecu-liarly, despite its apparent failure to re-locate to membranes,the GFP-Sch91–410-Akt1149–480 chimera displayed a p110�-dependent enhancement of phosphorylation at Thr-308 andSer-473 (Fig. 5C). This suggests that the N-terminal domain ofSch9may be sufficient to bring the chimera into proximity withyeast PDK1- and PDK2-like kinases, even though it may notretain the protein at the plasma membrane efficiently. Thus,stable recruitment at the plasma membrane seems to berequired to induce full toxicity of Akt in yeast. The antibodiesused proved to be specific for phosphorylated Akt1, failing todetect a putative phosphorylation on the conserved T-loop andHM residues of yeast Sch9 or the Akt1–148-Sch9411–824 chi-

FIGURE 3. All three Akt isoforms are activated in vivo by p110� inyeast. A, growth of the yeast WT strain (YPH499) is equally impaired byexpression of Akt1, Akt2, and Akt3 in the presence of the catalytic subunit ofPI3K. The three Akt isoforms were expressed from the corresponding pYES2-GFP-Akt vectors, and p110� from the YCpLG-myc-p110�. Media and growthconditions were as in Fig. 1A. �, means empty YCpLG vector. B, in vivo activa-tion of heterologous Akt isoforms in yeast. Lysates from the same transfor-mants as in A co-expressing GFP-Akt1, -Akt2, and -Akt3 with or withoutp110�, as indicated, were analyzed by immunoblotting with the antibodiesindicated: commercial phospho-specific anti-Akt1 antibodies directedagainst P-Thr-308 or P-Ser-473, anti-GFP antibodies, and anti-actin antibod-ies, as controls for equivalent protein loading.




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mera. Overall, these data support a very limited functional con-servation between Sch9 and Akt1 and essentially underscorethe importance of localization cues located in their N-terminalextensions for the function of these protein kinases.In Vivo Functional Studies of the PH Domain of Akt—To test

the utility of the yeast model to study the function of the PIP3-binding PH domain of Akt in vivo, we generated three pointmutations within this domain in Akt1: R25A, E40K, and E17K.The R25A and E40Kmutations had been previously reported torespectively eliminate and enhance the affinity of the PHdomain of Akt1 for PIP3 (12, 13). The E17K mutation wasrecently described as the first oncogenic mutation in PKB/Akt,and it also seems to enhance the affinity of the PH domain forlipids (20). Akt1R25A was not toxic for yeast cells, whereas theE40K and E17K mutations maintained toxicity when co-ex-pressed with p110� (Fig. 6A). Because interaction with PIP3 isimportant for the activation of the kinase, we investigated Akt1phosphorylation in thesemutants. In agreement with the effecton yeast growth observed, basal phosphorylation of Thr-308and Ser-473 was not enhanced in Akt1R25A by co-expression ofp110�, whereas Akt1E17K and Akt1E40K, like WT Akt1, didshow a PI3K-dependent enhancement of phosphorylation (Fig.6B). Quantification of the proportion of yeast cells in the pop-

ulation that re-locates GFP-Akt1 tothe plasma membrane provides asensitive indicator of PIP3 levels atthe yeast plasma membrane (23),so it should also serve as a readoutof the affinity of the Akt1 PHdomain to PIP3 in vivo. After 4 h ofco-expression in galactose-basedmedium, 60% of p110�-expressingcells had their membrane deco-rated by Akt1, whereas Akt1E17Kand Akt1E40K were present in themembranes of 80–90% of the cells(Fig. 6C). In contrast, Akt1R25Awas not re-located to the plasmamembrane in the same experi-mental conditions. These findingsconfirm in vivo that the R25Amutation precludes recognition ofPIP3, whereas both the E17K andE40K mutations enhance theaffinity of Akt1 for cellular mem-branes containing this lipid.Activated Akt Stimulates the CWI

MAPK Pathway Upstream the Rho-GTPase Exchange Factor Rom2—Tocheck whether phosphorylationevents elicited by active heterolo-gous Akt might influence signalingin the yeast cell, the phosphoryla-tion status of ERK-related yeastMAPKs was studied by using anti-phospho-ERK1/2 antibodies. Wefound that phosphorylation of theSlt2 MAPK, readily detectable with

these antibodies (37), was enhanced in lysates co-expressingp110� and Akt isoforms (Fig. 7A). In contrast, overexpressionof Sch9, either in the absence or in the presence of p110�, wasunable to trigger Slt2 phosphorylation (data not shown). Toprove that activation of the Slt2 MAPK pathway in these cellswas indeed dependent on the catalytic activity ofAkt andnot onthe presence of p110�, we comparatively examined lysatesfrom cells expressing Akt1 or Akt1K179M in the presence ofp110�. Slt2 phosphorylation was significantly higher in cellsexpressing catalytically active Akt1 than in cells expressing thekinase-dead version of the protein (Fig. 7B). To check whetherSlt2 phosphorylation depended on the activation of the MAPKmodule we expressed p110� and Akt1 in a bck1� background,lacking the MAPKKK at the head of the cell integrity pathway(Fig. 7C). As expected, deletion of bck1 precluded Akt-depend-ent Slt2 phosphorylation without affecting the basal activationlevels of other ERK-related kinases such as Kss1, also detectableby these anti-phospho-MAPK antibodies (Fig. 7C). This resultindicates that activated Akt stimulates the pathway upstreamthe MAPK module. Recruitment of protein kinase C (Pkc1) byGTP-bound Rho1 is known to be the key for the activation ofthe Bck1-Mkk1/2-Slt2MAPKmodule (see Fig. 7E). Rho1 func-tion is positively regulated by GTPase exchange factors and,

Glucose Galactosep110α Akt1

-+-+-+-+

WT

T308A

S473A

T308A,S473A

mAkt1 ITPPDQ--DDSMECVDSERRPHFPQFSYSASGTA mAkt2 ITPPDRY--DSLDPLELDQRTHFPQFSYSASIRE mAkt3 ITPPEKYDDDGMDGMDSERRPHFPQFSYSASGRE

HM

*

473

hAkt3-γ1 ITPPEKcqqsdcgmlgnwkk

467

anti-GFP

p110α - + - + - + - + - +

454

anti-P-Akt(T308)

Akt1 1-454WT F472AF469A

Y474A 1-467

WT T308A S473AT308AS473A

p110α - + - + - + - +WT T308D S473D

T308DS473DAkt1

anti-P-Akt(T308)anti-P-Akt(S473)

anti-GFP

p110α - + - + - + - +Akt1

anti-P-Akt(T308)anti-P-Akt(S473)

anti-GFP

Glucose Galactosep110α Akt1

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F469A, F472A

Y474A

1-467

1-454

FIGURE 4. Phosphorylation of Akt1 Thr-308, but not Ser-473 is required for toxicity in yeast. A, growthinhibition of the WT (YPH499) strain induced by WT Akt1 when co-expressed with p110� is attenuated by aT308A mutation, but not by S473A. The different Akt1 versions were expressed from the corresponding pYES3-GFP-Akt1 vectors, and p110� from the YCpLG-myc-p110� plasmid. Spotting on the appropriate selectivemedia was performed as in Fig. 1A. B, substitution of the Thr-308 phosphorylation site for Ala diminishes butdoes not preclude phosphorylation at Ser-473 and vice versa. Lysates from WT (YPH499) cells expressing theindicated GFP-Akt1 versions in the presence and absence of p110� as in A were immunoblotted with theindicated phospho-specific anti-Akt1 antibodies, and with anti-GFP antibodies as a control for equivalentprotein loading. C, phospho-mimetic mutations in either Thr-308 or Ser-473 do not influence phosphorylationof each other by yeast kinases. Experimental conditions were as in B. D, alignment of the C-terminal HM-containing extension of murine (m) isoforms of Akt and the human (h) alternatively spliced isoform of Akt3(Akt3-�1) showing the high degree of conservation at the hydrophobic motif (HM). Residues identical to themAkt1 sequence are highlighted in bold. In lowercase, the residues that derive from alternative splicing inhAkt3-�1. Multiple alignment was performed with ClustalW. The numbering corresponds to the mAkt1sequence. Arrows indicate the hydrophobic residues targeted in this study. The asterisk marks the Ser-473phosphorylatable site. E, mutational analyses on the C-terminal HM of Akt1. Lysates from WT (YPH499) cellsexpressing the indicated Akt1 WT or mutant versions from the corresponding pYES-GFP-Akt1 vectors in thepresence ((�);YCpLG-myc-p110�) or absence ((�); YCpLG) of p110� were spotted on agar media as in Fig. 1A.F, Akt1 HM mutations or truncations do not affect phosphorylation of Thr-308. Lysates from the same trans-formants as in E were analyzed by immunoblotting with the indicated antibodies.




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among them, Rom2 has a major involvement (38). Weexpressed p110� and Akt1 (or kinase-dead Akt1 as a negativecontrol) both in a rom2� mutant and in an isogenic wt strain.We could not detect Akt-dependent activation of the pathwayin the rom2� mutant, indicating that the Rom2 GTPaseexchange factor is required for the activation of the yeast CWIpathway in response to heterologous Akt1 activation (Fig. 7B).However, deletion of different CWI genes, such as ROM2,

BCK1, and SLT2, did not eliminate Akt-induced toxicity (Fig.7D), proving that this effect cannot be exclusively ascribed tothe activation of this pathway.CWI Activation Provides Biochemical and Transcriptional

Reporters for Heterologous Akt Function—We next testedwhether the phosphorylation levels of the Slt2 MAPK could berelated to the ability of Akt to become activated in yeast. Asshown in Fig. 7F, phosphorylation of Slt2 was dependent on thepresence of Thr-308 at the activation loop of Akt1 but did notrequire its HM C-terminal extension. Thus, CWI MAPK acti-vation by Akt1 matches its toxicity in yeast, as shown above in

FIGURE 5. Comparative analyses on Sch9, Akt1, and chimerical constructsthat interchange their regulatory and catalytic domains. A, growth of theWT (YPH499 strain) co-transformed with YCpLG (�) or YCpLG-myc-p110�(�), and pYES2 (Vector) or the same plasmid bearing GFP-Akt1, GFP-Sch9 orthe chimerical GFP fusions indicated. Media and growth conditions were as inFig. 1A. B, cellular localization of GFP-Sch9, GFP-Akt1, and the chimericalfusions. Transformants as in A were incubated in liquid synthetic mediumwith raffinose (2% w/v) as carbon source to mid-exponential phase, galactosewas then added up to 2% (w/v) for induction and incubation proceeded for4 – 6 h. Bars represent 5 �m. C, expression and analysis of the phosphorylationof Akt1, Sch9, and the chimerical proteins. Lysates from the same transfor-mants as above were processed and immunoblotted with the same antibod-ies and techniques as in Fig. 4F.

FIGURE 6. Yeast as a tool to evaluate the oncogenicity of Akt1. A, functionof the Akt1 PH domain is crucial for yeast growth inhibition. YPH499 transfor-mants harboring the pYES3-GFP-Akt1 (upper panel; WT) or pYES2-GFP-Akt1(lower panel; WT) plasmids, or the indicated mutants, and either YCpLG (�) orYCpLG-myc-p110� (�), were processed in the appropriated media as in Fig.1A. B, functionality of the PH domain determines Akt1 activation in yeast.Lysates from the same transformants as in A, grown in the appropriatedmedia were processed and immunoblotted with the same techniques andantibodies as in Fig. 4F. C, recruitment of GFP-Akt1 to yeast membranesreflects the affinity of its PH domain for PIP3. YPH499 transformants harboringYCpLG-p110� and pYES3-GFP-Akt1 or pYES2-GFP-Akt1 WT or mutant ver-sions as above were grown in the appropriated liquid synthetic medium withraffinose (2% w/v) as carbon source to mid-exponential phase, and expres-sion was induced by adding galactose up to 2% (w/v) and incubating for 4 h.Cultures were processed for fluorescence microscopy, and the percentage ofcells with GFP-Akt1 decorating the plasma membrane over the total of fluo-rescent cells was counted. Data are the average from three different transfor-mants processed in parallel. Error bars correspond to the standard deviation.p values were determined by Student’s t test.




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Fig. 4 (A and E). Also, Slt2 phospho-rylation was slightly more promi-nent in lysates expressing GFP-Akt1E40K and GFP-Akt1E17K in thepresence of p110� than in thoseexpressing WT GFP-Akt1 in thesame conditions (Fig. 7G), indicat-ing that the CWI pathway is sensi-tive to the efficiency of GFP-Aktlocalization to the plasma mem-brane via its PH domain. The factthat in vivo activation of Akt modu-latesMAPK signaling in yeast opensthe possibility of using transcrip-tional reporters to monitor Aktactivity. We used a construct bear-ing the promoter of theMLP1 gene,a major transcriptional target of theCWI pathway (39) coupled to lacZ,so that �-galactosidase activitycould be quantified (27). As shownin Fig. 7H, this system alloweddrawing the same conclusions asqualitatively observed above.Importantly, mutations in the PHdomain causing either loss or gain offunction, such as the oncogenicE17K, could be traced by thesemeans.Ala Scanning of the VL1 Region at

the PIP3-binding Pocket of Akt1 PHDomain—To further demonstratethe applicability of our model togaining insight into Akt functionand activation in vivo, we changedto Ala the amino acids in andaround the VL1 loop of the PHdomain of Akt1. This region config-ures a conserved structural ele-ment of the pocket that fits thepolar head group of PIP3 (11) (res-idues Arg-15 to Pro-24; Fig. 8A).In agreement with a key role of thisregion in PIP3 recognition and Aktfunction, 9 out of the 10 Ala-scan-ning mutants led to important(G16A) or severe loss of function,as judged by both failure of p110�-triggered membrane recruitmentand lack of toxicity in yeast (Fig.8B). The exception was Pro-24that, despite its vicinity to impor-tant residues, could be changed toAla without influence in the acti-vation or activity of the protein.This was not due to instability ofthe mutant protein, because allmutants were expressed in yeast to

FIGURE 7. Activation of the yeast CWI pathway provides a reporter for in vivo activation of heterologousAkt1. A, all three isoforms of Akt cause PIP3-dependent phosphorylation of the Slt2 MAPK. The same mem-branes as in Fig. 3B were re-hybridized with anti phospho-p44/p42 antibodies or anti-Slt2 antibodies, as acontrol for the amount of Slt2 in the lysates, as indicated. B, Slt2 activation requires an intact Akt1 kinase and thefunction of the ROM2 gene. Lysates from WT BY4741 S. cerevisiae strain or isogenic rom2� cells expressing Akt1(WT) or Akt1K179M (KD, kinase-dead) from the corresponding pYES2-GFP-Akt1 plasmid and p110� from theYCpLG-myc-p110� plasmid, as indicated, were immunoblotted with anti-phospho-p44/p42 antibodies, andanti-Slt2 antibodies. C, MAPK phosphorylation in yeast cells expressing in vivo-activated Akt1 is specific for Slt2and requires the upstream MAPKKK Bck1. Lysates from WT BY4741 and an isogenic bck1� mutant expressingAkt1 and p110� from the same vectors as above were analyzed by immunoblotting with anti-phospho-p44/p42 antibodies. D, deletions in essential components of the CWI MAPK pathway do not abrogate Akt-inducedtoxicity in yeast. BY4741 WT or isogenic rom2�, bck1�, and slt2� strains were co-transformed with YCpLG-p110�(H1047R) and pYES2-GFP (�) or pYES2-GFP-Akt1 (�) as indicated. Because the BY4741 strain is not assensitive as YPH499, used in the rest of the experiments in this report, to Akt1-induced toxicity, hyperactiveH1047R p110� mutant was used to enhance the phenotype. Experimental conditions were identical to thosein Fig. 1. E, scheme of the effects of in vivo Akt activation in yeast: Conversion of cellular PIP2 pools in the yeastcell to PIP3 by PI3K p110 catalytic subunit artificially recruits Akt to the plasma membrane, where phosphoryl-ation in both the T-loop (Thr-308) by PDK1 orthologs Pkh1 and Pkh2 (53) and the C-terminal HM (S473) by anunknown kinase (22) are enhanced. This leads to an activation of the Akt kinase that causes a rapamycin-inde-pendent negative effect on yeast growth as well as activation of the CWI MAPK signaling pathway that involves thefunction of the Rho1-GTPase exchange factor Rom2. Both effects require PIP3, T308 phosphorylation and Akt kinaseactivity, while phosphorylation of the HM is dispensable. F, Activation of the CWI pathway by different Akt1 mutants.Upper panel: Elimination of the Thr in the activation loop of Akt1 restrains its ability to activate the Slt2 MAPK. Lowerpanel: HM mutants or HM truncated versions of Akt1 are still able to induce PIP3-dependent Slt2 phosphorylation.The same membranes as in Fig. 4 (B and F) were hybridized with anti-P-p42/44 or anti-Slt2 antibodies as indicated.G, activation of the CWI MAPK Slt2 in the presence of Akt1 loss- and gain-of-function PH mutants. The same mem-branes as in Fig. 6B were re-hybridized with anti phospho-p44/p42 antibodies or anti-Slt2 antibodies, as indicated.H, quantification of Akt1 activity in vivo in the yeast cell using a pMLP1-lacZ CWI transcriptional reporter. YPH499 cellswere co-transformed with YCpLG-p110�, and pYES2-GFP-Akt or pYES3-GFP-Akt WT or mutant versions as above,and the pMLP1-LACZ(URA3) or pMLP1-LACZ(HIS3) plasmids. Cells were grown in synthetic raffinose-based mediumlacking uracil, leucine, and histidine or tryptophan, as required, induced by the addition of galactose for 6 h, and theexpression of MLP1 promoter-driven lacZ was examined. �-Galactosidase activity is expressed in the y-axis as nano-moles of o-nitrophenyl-�-D-galactopyranoside converted/minute/mg of protein. Data are the average from tripli-cate experiments on independent yeast transformants (except WT, six replicas). p values were calculated by Stu-dent’s t test. Error bars correspond to the �S.D.




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the same levels of as WT Akt1, as judged by immunoblot onyeast lysates (data not shown).The VL1 loop includes Glu-17, whose change to Lys yielded

the above-mentioned gain-of-function oncogenic Akt1allele (20). Other PH domains, such as that of Bruton’s tyro-sine kinase, include VL1 extensions rich in basic residues(40). Positively charged residues in this region provide a sur-face that could interact with negatively charged lipid mem-branes as well as facilitate interaction with the phosphategroups of phosphoinositides. From the DNA sequence ofmurine Akt1-coding DNA, we searched for feasible singlepoint mutations that change any of the residues at the VL1region to basic polar amino acids. Besides E17K (GAA toAAA), we found two other possible mutations in codonsconserved in the human sequence at the DNA level: G16R(GGG to CGG) and P24R (CCA to CGA). Thus, we wereprompted to study whether these mutations would also alterAkt1 PIP3 recognition and function. As shown in Fig. 8B,G16R maintained toxicity and led to a more efficient recruit-ment to PIP3-enriched membranes than G16A, although lessthan WT Akt1. Interestingly, the P24R mutant, a substitu-tion that naturally exists in other PH domains, like that of thegeneral receptor for phosphoinositides (GRP1 (41)), was

slightly but reproducibly more responsive to PIP3 than WTAkt1 (Fig. 8B). These data underscore the sensitivity of theyeast system proposed here to study both loss- and gain-of-function mutants of Akt.

DISCUSSION

Humanized yeast systems are valuable tools to performgenetic and pharmacological screens on heterologous genes orproteins (42, 43). Such systemsmay take advantage on the con-servation of pathways from yeast to mammals, allowingcomplementation of the function in yeast by the human gene;or, alternatively, expression of heterologous proteins may pro-vide functions that do not naturally exist in yeast. Based on ourformer observations that mammalian Akt1 could be re-locatedto membranes and phosphorylated in a PI3K-dependent fash-ion in S. cerevisiae (22), we report here that in vivo activation ofthe different mammalian Akt isoforms in the yeast model leadsto growth inhibition, an effect that relies on its catalytic activity.We propose this system as a valuable tool to evaluate in vivo thefunctional effects of Akt mutations, which may be relevant forstudies on oncogenesis.PIP3-dependent activation of Akt led to the activation of the

CWI MAPK pathway that lies downstream the Rho1 GTPaseand the protein kinase C yeast homolog Pkc1. Although hyper-activation of this pathway is known tonegatively influence yeastgrowth (44), we show thatAkt-induced Slt2 activation is not thecause of toxicity in yeast. We demonstrate that the Rho1 GAPRom2 is required for activation of the pathway in yeast cellsco-expressing PI3K and Akt. To know whether heterologousAkt directly phosphorylates Rom2 or operates on componentsupstream in the pathway will require further experimentation.We cannot discard that Akt phosphorylation is somehow alter-ing the properties of the cell wall, and the CWI pathway issubsequently activated. Actually, electron microscopy on yeastcells expressing activated Akt show abnormal cell walls.4Regardless of themechanism involved, the activation of the Slt2MAPK route provides the possibility of using a quantitativereporter of the function of Akt in an in vivo context.Finding the heterologous targets of activated Akt in yeast

might reveal novel conserved Akt substrates in the future. Thefact that the Tor inhibitor rapamycin does not interfere withAkt toxicity indicates that TORC1 activation is not related toAkt1-induced toxicity in yeast. Accordingly, no regulation ofyeast TORC1 by AGC kinases, such as the putative Aktortholog Sch9, has been reported to our knowledge. Rather, ithas been demonstrated that Sch9 acts downstream Tor1 (45),yielding it a more likely functional ortholog to the mTor targetS6K than to Akt. In this regard, we show here that, in the pres-ence of PI3K, Sch9 overexpression does not lead to the samegrowth inhibitory effects than Akt. Also, exchanging the N-ter-minal membrane-binding domains of Akt1 and Sch9 allowedmimicking the Akt phenotype only partially, even though PIP3-dependent membrane localization of the Sch9 C-terminalkinase domain mediated by the Akt1 N-terminal PH domainwas as efficient as that of Akt1. We have observed, however,

4 I. Rodríguez-Escudero, A. Andres-Pons, R. Pulido, M. Molina, and V. J. Cid,unpublished data.

FIGURE 8. Mutational and functional analysis of the VL1 region of Akt1.A, alignment of the 30 N-terminal residues of hAkt1 and mAkt1 with mAkt2and mAkt3. Shaded boxes indicate identical residues taking mAkt1 as a refer-ence. The image at the top shows the secondary structure in Akt1 associatedto the sequences below. The region selected for the Ala-scanning mutationalanalysis is underlined. Asterisks mark residues previously mutated to alanineand reported to be essential (11). The black arrowhead indicates the residueGlu-17, in which the E17K oncogenic mutation maps. Gray arrowheads pointthe residues that have been mutated to Arg in this work. B, functional effectsof Akt1 mutations at the VL1 region. Localization of GFP-Akt1 to membranesin the presence of PI3K was analyzed after 4 h of induction in galactose as inFig. 6C. Data are the average from three different transformants processed inparallel. Error bars correspond to the standard deviation. p values were calcu-lated by Student’s t test (upper panel). Spotting on the appropriate selectivemedia was performed as in Fig. 1A (lower panel; controls in glucose grewequivalently and are not shown).




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that overexpression of Akt isoforms by themselves, in theabsence of PI3K, cause a very mild growth inhibition that isequivalent to the one observed when Sch9 is overexpressed, sowe cannot discard that both kinases could share substrates.Howmight the yeast model contribute to our understanding

of the mechanisms that lead to Akt activation? Some specula-tions can be driven from our observations in yeast. First, it isintriguing that myr-Akt1, widely used as a constitutively acti-vable version of Akt that bypasses the need for PI3K, is not toxicby itself in yeast. This suggested that PIP3 binding of Akt andnot its sheer recruitment to membranes is critical for its activa-tion, in agreement with previous structural studies (46) and invivo Forster resonance energy transfer experiments (47). Analternative explanation to the failure of myr-Akt to behave asconstitutively active in S. cerevisiae could be that recognition ofendogenous Akt targets involved in the slow growth phenotypeobserved may rely on detachment of activated Akt from mem-branes, a process that myristoylation should prevent. Second,we can address the controversial questionwhether phosphoryl-ation of Thr-308 conditions Ser-473 phosphorylation and viceversa. Our results with T308A and S473A mutants support theidea that phosphorylation of one of the residues is conforma-tionally favorable, but not essential, for the phosphorylation ofthe other. On the other hand, the fact, that phosphorylation ofeither residue still occurs and is responsive to PIP3 when theother phosphorylatable residue is eliminated, is in agreementwith reports that revealed that Thr-308 phosphorylation wasunaffected in cells ablated for mTORC2 activity, the purportedSer-473 kinase (48), as well as with those showing that cellsdefective in PDK1 still phosphorylate Ser-473 (49). Third, Ser-473 phosphorylation, and even thewholeHM, is largely dispen-sable for the growth inhibitory effects of Akt in yeast. This wassurprising, considering the facts that Ser-473 phosphorylationis regarded as an important event for full activation of thekinase and that such residue is phosphorylated by yeast endog-enous kinases. However, if the effects of Akt in yeast wouldmostly depend on the phosphorylation of local submembranetargets, these results are in agreement with the emerging viewthat Thr-308 phosphorylation is sufficient for membrane-bound Akt1 to efficiently phosphorylate local targets, whereasSer-473 phosphorylation would be required for activity of thekinase on non-submembrane targets, such as nuclear sub-strates (50).The best support for the value of our yeast model arises from

functional analyses of Akt1 mutated at its PH domain. In ourAla scanning of the VL1 region and surrounding amino acids,which contributes to the structure of the phosphoinositidebinding pocket, we prove that these residues are either essential(Arg-15, Glu-17, Tyr-18, Ile-19, Lys-20, Thr-21, Trp-22, andArg-23) or very important (Gly-16) for the function of the PHdomain. Mutations to Ala of Lys-14 within the �1 strand orArg-25 within the �2 strand, the residues that flank the regiontargeted here, had also been reported to yield versions of Aktunable to bind PIP3, and thus non-activable (11). At least in thecase of theR25Amutant, we also prove that yeast reproduces itsloss of function. Remarkably, the only oncogenic mutationdescribed to date in Akt1maps to theGlu residue at position 17(20), within the VL1 region, and involves its change into a basic

residue. Expression in yeast of this allele in the presence of PI3Kleads to an enhanced phenotype, as evidenced by a greater relo-cation of the mutant protein to cellular membranes. This is animportant observation, because it proves that yeast is sensitivenot only to loss-of-function but also to gain-of-function Aktmutants, allowing the use of this system to analyze putativeoncogenic alleles. The E40K allele, which introduces a positivecharge in the membrane-interacting interface of the proteinoutside the PIP3 binding pocket (13), also showed enhancedphenotypes in PIP3-producing yeast, although this mutationhas not been related to tumorigenesis. We have also generated anovel point mutation leading to a P24R substitution that yieldedAkt1 slightly more responsive to PIP3, as judged by membranerelocation of GFP-Akt1. This result suggests that the addition ofpositive charges at positions other thanGlu-17 at the PIP3 bindingpocket could enhance affinity for lipids. TheP24Rmutation, how-ever, unlike Akt1E17K (20), did not lead to enhanced Thr-308 orSer-473Akt1phosphorylationwhen expressed inmammalian celllines.5 This indicates that it may not be relevant as an oncogenicallele, as suggestedby the fact that no clinical reports are describedinvolving this particular mutation.In sum, our data illustrate the value of the humanized yeast

model to readily study Akt structure-function relations. In thiscontext, the development of qualitative or semi-quantitativevisual readouts (such as yeast growth, GFP-Akt membranerelocation, or Slt2 MAPK phosphorylation), as well as reportersystems specific for Akt activation in yeast, such as theMLP1-lacZ described here, provide novel tools that could be furtherexploited in the future. For example, othermutations predictedto enhance the affinity for PIP3 of PH domains, such as thoserecently suggested by bioinformatic means by Park andco-workers (51), could be tested using this model. Besidesgenetic analyses, the system might also be utilized to test orscreen for small molecules that display Akt inhibitory proper-ties or, alternatively, to screen for mutations that confer resist-ance to known inhibitors. The latter strategy has recently beenperformed on PI3K quite successfully (52), proving that yeast-based systems can be a very helpful tool for the molecular anal-yses of key pharmacological targets, such as PKB/Akt.

Acknowledgments—We thank T. M. Badger, A. B. Sanz, R. García,and J. Arroyo for plasmids, J. Ortiz-Rincon for his help, and J. W.Thorner for critical comments on the manuscript.

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Víctor J. CidIsabel Rodríguez-Escudero, Amparo Andrés-Pons, Rafael Pulido, María Molina and

Akt Mutations Model for the Functional Study ofin Vivo, an Saccharomyces cerevisiaeB/Akt in

Phosphatidylinositol 3-Kinase-dependent Activation of Mammalian Protein Kinase

doi: 10.1074/jbc.M807867200 originally published online March 23, 20092009, 284:13373-13383.J. Biol. Chem.

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