Advances in Enzyme Regulation 52 (2012) 46–57

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advenzreg

Role of the C-terminal regulatory domain in the allostericinhibition of PKB/Akt

Véronique Calleja a,**, Michel Laguerre b, Banafshé Larijani a,*aCell Biophysics Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, WC2A 3LY London, UKb IECB-CBMN-UMR 5248-CNRS, Institut Européen de Chimie et Biologie, Université de Bordeaux, 2 rue Robert Escarpit,33607 Pessac Cedex, France

Introduction

Protein kinase B (PKB/Akt) (EC¼ 2.7.11.1) is a soluble serine/threonine kinase that belongs to the AGCsuper family. It has been shown to have a critical role in key aspects of cell signaling, metabolism andsurvival and has been found to be dysregulated in cancer (for review (Manning and Cantley, 2007;Restuccia and Hemmings, 2010)). PKB/Akt is activated by binding to phosphoinositides (PtdIns(3,4)P2and PtdIns(3,4,5)P3) at the plasma membrane (Andjelkovic et al., 1997), and through phosphorylation ofThr308 and Ser473 (numbering for PKBa/Akt1) (Alessi et al., 1996) by its upstream activators,phosphoinositide-dependent protein kinase 1 (PDK1) (EC¼ 2.7.11.1) (Alessi et al., 1997) and mammaliantarget of rapamycin complex 2 (mTORC2) (EC ¼ 2.7.11.1) (Sarbassov et al., 2005) respectively. In the lastdecade great efforts have been dedicated to finding ways of inhibiting PKB/Akt with high specificity.Merck developed a series of allosteric small molecule inhibitors (non ATP-competitive) that only tar-geted PKB/Akt when the PH (pleckstrin homology) domain was present, explaining the high specificityas compared to other related AGC kinases (Lindsley et al., 2005; Zhao et al., 2005). Inhibition of the kinaseactivity was accompanied by the loss of phosphorylation of the regulatory residues Thr308 and Ser473(Barnett et al., 2005). The first commercialized inhibitor of this series, Akt inhibitor VIII (also known asdual Akt1/2 inhibitor), was shown to be selective for PKBa/Akt1 (IC50 58 nM) and PKBb/Akt2 (IC50210 nM) compared to PKBg/Akt3 (IC50 2119 nM) (Lindsley et al., 2005). Recently the next generationallosteric compound derived from Akt inhibitor VIII (MK-2206) was successfully used in a phase I clinicaltrial (Lindsley, 2010). Our group and others had previously proposed that Akt inhibitor VIII was bindingat the interface of the PH and kinase domains (Barnett et al., 2005; Calleja et al., 2009) and we furthershowed that this positioning locked PKB/Akt in the PH-in inactive conformation, explaining the allostericmechanism of inhibition and the lack of phosphorylation of Thr308 (Calleja et al., 2009). Recently, the co-crystal structure of PKBa/Akt1 with Akt inhibitor VIII was published, showing a binding site dependent

* Corresponding author. Tel.: þ44 207 269 3082; fax: þ44 207 269 3094.** Corresponding author. Tel.: þ44 207 269 3054; fax: þ44 207 269 3094.

E-mail addresses: veronique.calleja@cancer.org.uk (V. Calleja), banafshe.larijani@cancer.org.uk (B. Larijani).

0065-2571/$ – see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.advenzreg.2011.09.009

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on the PH and the kinase domains of PKBa/Akt1 and confirming that inhibitor binding maintained theprotein in its inactive conformation (Wu et al., 2010). However in absence of the C-terminal domain fromthe co-crystal structure, the effect of the inhibitor on Ser473 could not been explained. In an earlier studywe showed by mutagenesis that the binding site for the inhibitor in PKBa/Akt1 kinase domainwas closeto, or overlapping in part with, the binding site for the C-terminal regulatory domain. In addition weshowed that the presence of the inhibitor prevented the phosphorylation of the C-terminal regulatoryresidue Ser473 by mTORC2 (Calleja et al., 2009).

In the present study we have used molecular modeling and dynamics combined with mutagenesisand classical biochemistry to evaluate the impact of key amino acid residues of PKBa/Akt1 and PKBg/Akt3 on the binding to Akt inhibitor VIII. Our experiments were guided by predictions that could bemade from the co-crystal structure (Wu et al., 2010) and from our previously published dynamicmodelof the inactive conformer of PKBa/Akt1 (Calleja et al., 2009). Our results suggest that the C-terminalregulatory domain of PKB/Akt is likely to play an important role in the positioning of Akt inhibitor VIIIin vivo and that the absence of the C-terminal regulatory domain in the co-crystal structure might havepotentially led to an alteration of the inhibitor’s position.

Material and methods

Akt inhibitor VIII was from Calbiochem (Merck KGaA). Human PDGF was from R&D Systems. Rabbitmonoclonal phospho-Akt Thr-308 and Ser-473 were from Cell Signaling (New England BioLabs UK).The anti-GFP antibodywas an in-housemousemonoclonal. Other chemicals were from Sigma–Aldrich.The Odyssey blocking buffer was from LI-COR UK. The IRdye 800CW anti-rabbit and IRdye 680LT anti-mouse were used as secondary antibodies for the LI-COR/Odyssey system (from LI-COR). Lipofect-AMINE/PLUS reagent and transfection medium OPTIMEM were from GIBCO-BRL.

DNA constructs and direct site mutagenesis

pEGFP-HA Akt construct was described previously (murine AKT1; Genbank accession numberNM_009652) (Watton and Downward, 1999); we called this construct GFP-PKBa/Akt1 in this article.

GFP-PKBg/Akt3 was created by PCR amplification of PKBg/Akt3 from pCDNA3-Myr HA Akt3 human(Addgene plasmid 9017). The oligonucleotides used for the amplification were: PCR-PKBg sense: gtcgac atg tac cca tac gat gtt cca gat tac gct tcc aga atg agc gat gtt acc att gtg aaa gaa ggt tgg; and PCR-PKBgantisense: gga tcc tta ttc tcg tcc act tgc aga gta gga aaa ttg agg g. Prior to the PCR amplification of PKBg,an internal BamHI site was removed in pCDNA3-Myr HA Akt3 by direct site mutagenesis using: PKBg-BamHI-less sense: ctt tca ggg ctc ttg ata aag gac cca aat aaa cgc ctt gg; and PKBg-BamHI-less antisense:cca agg cgt tta ttt ggg tcc ttt atc aag agc cct gaa ag. The PCR product was subcloned into the intermediatecloning vector zero blunt TOPO from Invitrogen, then excised using SalI-BamHI restriction enzymes.The excised PCR amplificationwas then put in phase in C-terminus of GFP (instead of PKBa in the vectorGFP-PKBa cut with SalI-BamHI.

The mutants of PKB/Akt were obtained by direct site mutagenesis using the Quick Change muta-genesis kit from Stratagene with the oligonucleotides:

PKBa-S205A sense: cgt gtc ctg cag aac gct agg cat ccc ttc c; and PKBa-S205A anti: gga agg gat gcc tagcgt tct gca gga cac g;

PKBa-Q203K sense: ctt act gag aac cgt gtc ctg aag aac tct agg cat ccc ttc; and PKBa-Q203K anti: gaaggg atg cct aga gtt ctt cag gac acg gtt ctc agt aag;

PKBa-EKN-GK sense: gcc ctg gac tac ttg cac tcc ggg aag gtg gtg tac cgg gac ctg aag ctg; and PKBa-EKN-GK anti: cag ctt cag gtc ccg gta cac cac ctt ccc gga gtg caa gta gtc cag ggc;

PKBg-T203S sense: ctg aaa gca gag tat taa aga aca gta gac atc cct ttt taa cat cc; and PKBg-T203S-anti:gga tgt taa aaa ggg atg tct act gtt ctt taa tac tct gct ttc ag;

Cell culture and transfections

NIH3T3 cells from ATCC were maintained in DMEM supplemented with 10% Donor Calf Serum(DCS) and seeded at 150,000 in a well of a six-well plate. The transfections were done with 1 mg of DNA

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of the different constructs using LipofectAMINE/PLUS reagent in OPTIMEM medium as recommendedby the manufacturer (GIBCO-BRL). The cells were left for 3 h in the transfection mix, the medium wasthen removed and replaced with DMEM 10% DCS. The experiments were performed 24 or 48 h aftertransfection.

Cell lysis and western blot (LI-COR/Odyssey fluorescence system)

After stimulation or treatment as indicated, the cells were lysed for 15 min on ice in lysis buffer(20 mM Tris–HCl [pH 7.4], 150 mM NaCl, 100 mM NaF, 10 mM Na4P2O7, 10 mM EDTA supplementedwith COMPLETE protease inhibitor cocktail tablet (Roche)). To terminate the reaction, 4x SDS samplebuffer (125 mM Tris–HCl [pH 6.8], 6% SDS, 20% glycerol, 0.02% bromophenol blue supplemented with10% b-mercaptoethanol) was added, and the samples boiled for 5 min. The proteins were separated ona 10% SDS-PAGE gel.

After electrophoresis, the polyacrilamide gels were transferred onto PVDF membrane (ImmobilonFL; Millipore), incubated in the Odyssey blocking buffer for 1 h, and then with the different antibodiesphospho-Akt (Thr-308) antibody (rabbit monoclonal) or phospho-Akt (Ser-473) (rabbit monoclonalantibody) at 1:1000 together with an anti-GFP in-house mouse monoclonal at 1:2500 for 4 h inOdyssey blocking buffer. The membranes were then washed in PBS 0.2% Tween-20 with 1% odysseybuffer for 1 h. The secondary antibodies IRdye 800CWanti-rabbit and IRdye 680LT anti-mouse (LI-COR)were used at 1:5,000, in Odyssey blocking for 1 h. The blots were scanned with the infrared LI-CORscanner, allowing for simultaneous detection of two targets (anti-phospho and total protein) in thesame experiment. For each experiment, the values of the phospho bands were normalized for the totalamount of protein in each lane.

Molecular modeling and dynamics

Docking experiments were performed using VINA (Trott and Olson, 2010), starting from the modelstructure of the complex kinase þ PH (without the C-terminus). Three experiments were performedwith 3 different exhaustiveness parameters (8, 64 and 512). The 3 experiments are highly convergentand all show that even if the water cavity is coming from molecular dynamics and thus maybe notoptimized, the cavity is large enough to easily accommodate an aromatic moiety coming from oneextremity of the Akt inhibitor VIII. In fact either the tricyclic part or the imidazolidinone moieties areperfectly able to embed within the cavity. When the imidazolidinone part is within the cavity, thedistance between the closest aromatic rings is around 5.0–5.5 Å (s–p stacking), the carbonyl group ofthe inhibitor is very close to the NH of the highly conserved His194, and in some cases the NHþ group ofthe inhibitor is in close proximity to the lateral carbonyl of Gln218 (2.51 Å) or Gln203. In the first casethe tricyclic aromatic ring experiences a s/p–p stacking with His220 and in the second case there isa close interaction with Lys268. Otherwise, when the tricyclic ring is within the water channel, thenthere is also an s-p stacking with Trp80 and the NHþ group of the inhibitor is at very short distancefrom the lateral carbonyl of Gln218 or Gln203.

Molecular dynamics of the co-crystal structure with Akt inhibitor VIII was performed using GRO-MACS 4.5 software and Gromos 96 parameters (43A1). The PDB file 3O96 was used as a starting point.The force-field parameters for Akt inhibitor VIII were generated from the PRODRG server (Schuttelkopfand van Aalten, 2004). The protein complex was embedded in a cubic water box (of 100 Å side) fullyneutralized with 150 mM salt added. Afterward a classical molecular dynamics run was performed atconstant pressure (P ¼ 1 bar) and temperature (T ¼ 300 K) with a time step of 2 fs (LINCS was on).

Results and discussion

Based on the co-crystal structure of PKBa/Akt1 with Akt inhibitor VIII, several amino acid residueswere predicted to be important for the inhibitor binding. In order to examine whether these residuesplayed a significant role in vivo, we performed a series of mutations on GFP-tagged PKB/Akt constructs.Ser205 was predicted to be the only residue forming a direct hydrogen bond (H-bond) with Aktinhibitor VIII (Fig. 1A). The equivalent residue being a threonine in PKBg/Akt3 it could also partly

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explain the selectivity for PKBa/Akt1 compared to PKBg/Akt3 due to the weakening of this H-bond inthe latter (Fig. 1B). Therefore Ser205 was mutated to an alanine in GFP-PKBa/Akt1. Previous studies hadshown that the binding of Akt inhibitor VIII not only inhibited the kinase activity of PKB/Akt but alsoconcomitantly prevented the phosphorylation of its two regulatory residues Thr308 and Ser473.Therefore the phosphorylation state of these residues was used as a reporter for the sensitivity of PKB/Akt to the inhibitor. NIH 3T3 cells expressing GFP-PKBa/Akt1 wild type (AKT1) or mutated on theSer205 (AKT1-S205A) (Fig. 1C) were pre-treated for 30 min with increasing doses of Akt inhibitor VIII,as indicated, before stimulation with 30 ng/ml of PDGF for 5 min. The graphs present the level ofThr308 (left) and Ser473 (right) phosphorylations at different inhibitor concentrations, normalized forthe maximum phosphorylation obtained with PDGF stimulation in the absence of inhibitor. The datashow that the decrease in phosphorylation of Thr308 (left graph) and Ser473 (right graph) due toincreasing doses of inhibitor is not modified by the mutation S205A, indicating that the loss of thisresidue in themutant does not affect the sensitivity of PKBa/Akt1 for Akt inhibitor VIII. Alternativelywewanted to testwhether the reversemutation could render PKBg/Akt3more sensitive to the inhibitor bypotentially restoring a stronger H-bond. Therefore we mutated Thr203 for a serine on GFP-PKBg/Akt3(Fig. 1D). As expected, PKBg/Akt3 is less sensitive to Akt inhibitor VIII than PKBa/Akt1 with only 80%inhibition of the Thr308 phosphorylation and 60% inhibition of the Ser473 phosphorylation at 5 mMinhibitor, compared to a complete inhibition in PKBa/Akt1 (Fig. 1C). However the mutation T203S doesnot significantly change the sensitivity of GFP-PKBg/Akt3 for the inhibitor. Taken together these resultssuggested that the residue Ser205 did not contribute to the sensitivity of PKBa/Akt1 for Akt inhibitorVIII in cells. We also performed a 6 ns molecular dynamics simulation on the co-crystal structurereconstituted with the missing amino acids and containing the inhibitor (see Materials and methods)(Fig. 1E). Within 1 ns the distance between Ser205 and the inhibitor increased to 5 Å, and this distancewas maintained for the remainder of the simulation. Taken together these results suggest further thatSer205 was not an important residue for the binding of Akt inhibitor VIII in vivo, unlike what has beenproposed by Wu and collaborators.

From the co-crystal structure other potential residues were predicted to stabilize the inhibitor in thepocket formed at the interface between the PH and kinase domains of PKBa/Akt1. In particular, Trp80 ofPKBa/Akt1 PH domain was found to form a ring–stacking interaction with Akt inhibitor VIII (Figs. 1Aand 2A). This is in agreement with work from our group and others, which had previously shown thatthe mutation of the Trp80 to alanine induced a total loss of sensitivity to the inhibitor (Calleja et al.,2009; Green et al., 2008). Additionally a three residue turn (Glu267-Lys268-Asn269) in the kinasedomain of PKBa/Akt1 (shown in blue, Fig. 2A) was found to form polar interactions on the opposite sideof the inhibitor from the Trp80. Interestingly, the shortening of this amino acid turn in PKBg/Akt3 thatis replaced by the residues Gly–Lys (Fig. 2B), could possibly explain the difference in sensitivity of thetwo isoforms. Therefore wemutated GFP-PKBa/Akt1, replacing the Glu267-Lys268-Asn269 loop by theresidues Gly–Lys found in PKBg/Akt3 (mutant AKT1-EKN-GK). NIH 3T3 cells expressing GFP-PKBa/Akt1wild type (AKT1) or mutant (AKT1-EKN-GK) were pre-treated for 30 min with increasing doses of Aktinhibitor VIII, as indicated, before stimulation with 30 ng/ml of PDGF for 5 min (Fig. 2C). At suboptimalconcentration of inhibitor (1 mM and 2.5 mM of Akt inhibitor VIII) the phosphorylation of Thr308 (leftgraph) and Ser473 (right graph) was increased in the mutant AKT1-EKN-GK compared to the wild typeAKT1. At 1 mM the decrease in Thr308 phosphorylation went from approximately 75% in AKT1 to 50%(p ¼ 0.04, n ¼ 3) in the mutant AKT1-EKN-GK. There was a comparable decrease in Ser473 phos-phorylation (approximately 70% in AKT1 to 50% (p ¼ 0.03, n ¼ 3) in the mutant AKT1-EKN-GK).Similarly, the inhibition of Thr308 and Ser473 phosphorylation at 2.5 mM of inhibitor was significantlyreduced in the mutant (p ¼ 0.02 in both cases, n ¼ 3) compared to the wild type.

These results indicated that the mutant AKT1-EKN-GK was less sensitive than the wild typeAKT1 at suboptimal concentration of Akt inhibitor VIII. However it is interesting to note that theloss of the residues Glu267-Lys268-Asn269 did not totally prevent the inhibition of PKBa/Akt1since the phosphorylation of Thr308 and Ser473 was almost completely suppressed at 5 mMinhibitor. By performing a 12 ns molecular dynamics simulation (Fig. 2D) on the co-crystal structurereconstituted with the missing amino acids and containing the inhibitor (see Materials andmethods), we saw that the Lys268 was maintained in very close proximity (less than 5 Å) to Aktinhibitor VIII. This result suggests that Lys268 would be an important residue involved in the

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Fig. 1. Role of PKBa/Akt1 Ser205 and of the equivalent PKBg/Akt3 Thr203 in Akt inhibitor VIII binding. A) Image of the co-crystalstructure of the PH (pink) and kinase (green) domains of PKBa/Akt1 with Akt inhibitor VIII (yellow sticks). The Trp80 residuefrom the PH domain is in red. The Ser205 (gold) is within binding distance of the inhibitor. B) Alignment of the amino acid sequenceof PKBa/Akt1 and PKBgAkt3 from the residues 201 to 210 of PKBa/Akt1. Residue Ser205 is a threonine in PKBgAkt3. C) NIH 3T3 cellsexpressing GFP-PKBa/Akt1 (AKT1) or a mutant in which Ser205 was changed to an Ala (S205A) were treated for 30 min with

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stabilization of Akt inhibitor VIII in the region of the amino acid turn. This lysine residue beingconserved in the mutant AKT1-EKN-GK might explain why the loss of sensitivity is only partial andat suboptimal doses.

Since the co-crystal structure of PKBa/Akt1 with Akt inhibitor VIII did not contain the last 37residues of PKBa/Akt1 we opted to test whether the C-terminal regulatory domain of PKBa/Akt1 mighthave a role in the inhibitor binding. To do so we performed mutagenesis and biochemical experimentsto test the predictions derived from our molecular modeling and dynamics simulations. First, wedetermined what would be the possible binding site for Akt inhibitor VIII in accordance with ourpreviously published model, which shows that inhibition was the result of locking the inactive PH-inconformation of PKBa/Akt1. The docking of Akt inhibitor VIII with PKBa/Akt1 in the inactive PH-inconformation (previously obtained using dynamic simulations (Calleja et al., 2009)) is shown inFig. 3A. The Trp80 residue (red) from the PH domain is visible at the bottom of the cavity that it hasshaped by enforcing into the kinase domain of PKBa/Akt1. Several docking possibilities were obtainedwhere the imidazoquinoxaline or the benzimidazolonemoieties of the inhibitor could easily fit into thecavity (see Materials and methods). However the comparison of the chemical structures of Aktinhibitor VIII and the derived compound MK-2206 (Fig. 3B) suggested that the critical part of theinhibitor was the imidazoquinoxaline (see the superposition of the two inhibitors on themerged image– right panel). Therefore it was very likely that this moiety would be the one bound to the criticalresidue Trp80 locking the protein in its inactive conformation. In this position the residues Gln218(magenta) and Gln203 (dark purple) from the kinase domain would be at binding distance from theinhibitor (Fig. 3A). In particular Gln203 would be predicted to make an H-bond with the piperidiniumof Akt inhibitor VIII in this position. By comparison of the amino acid sequence of the three PKB/Aktisoforms (Fig. 3C) we observed that the residue corresponding to Gln203 in PKBa/Akt1 and PKBb/Akt2was a Lysine in PKBg/Akt3. The replacement of Gln203 with a lysine would be predicted to modify thepositioning of Akt inhibitor VIII by preventing the formation of an H-bond and exerting repulsiveelectrostatic interactions. This could explain in part the lower sensitivity of PKBg/Akt3 for the inhibitorcompared to PKBa/Akt1. To test this hypothesis, Gln203 was mutated to a lysine in PKBa/Akt1. NIH 3T3cells expressing GFP-PKBa/Akt1 wild type (AKT1) or mutated on the Gln 203 (AKT1-Q203K) were pre-treated for 30 min with increasing doses of Akt inhibitor VIII, as indicated, before stimulation with30 ng/ml of PDGF for 5 min (Fig. 3D). The graphs present the level of phosphorylation of Thr308 (left)and Ser473 (right) at different concentration of inhibitor, normalized for the maximum phosphory-lation obtained with PDGF stimulation in absence of inhibitor. Phosphorylation of Thr308 was totallyinhibited in the mutant AKT1-Q203K as in the wild type AKT1. This result implied that the binding ofthe inhibitor in the mutant protein induced the locking of the PH-in conformation as in the wild typePKBa/Akt1. However, unlike Thr308 the inhibition of the Ser473 phosphorylation was reduced in themutant AKT1-Q203K. At 1.5 mM of Akt inhibitor VIII the Ser473 phosphorylation went from approxi-mately 70% (AKT1) to 45% inhibition (AKT1-Q203K) (p¼ 0.008, n ¼ 6) and at 2 mM from approximately90% (AKT1) to 50% inhibition (AKT1-Q203K) (p¼ 0.001, n¼ 6). These results indicated that the effect ofthe inhibitor on the Thr308 and the Ser473 could be dissociated, thus that the mechanisms involved inthe inhibition of these two sites were partly independent. In our previous study we had determined(using the phosphatase inhibitor for PP2A, okadaic acid) that the loss of the Ser473 phosphorylation inthe presence of Akt inhibitor VIII was due to the lack of phosphorylation by mTORC2 rather than anincreased dephosphorylation of this residue (Calleja et al., 2009) indicating that the phosphorylation ofSer473 was likely to be prevented by steric hindrance. The results of these experiments are in agree-ment with our model since the loss of an H-bond between Gln203 and the NHþ of the inhibitor in the

different concentrations of Akt inhibitor VIII as indicated prior to 5 min PDGF stimulation. Thr308 (left) and Ser473 (right) phos-phorylations were detected by western blot using phospho-specific antibodies, and bands were quantified using the Odyssey/LI-CORfluorescence detection system. The data are presented as a percentage of Thr308 or Ser473 phosphorylations upon maximum PDGFstimulation (5 min) for each construct. D) NIH 3T3 cells expressing GFP-PKBg/Akt3 (AKT3) or a mutant in which Thr203 was changedto a serine (T203S) were treated as in C). Thr308 (left) and Ser473 (right) phosphorylations were detected by western blot usingphospho-specific antibodies. The quantifications and data presentation were as in C). E) The graph shows the distance between theSer205 and the inhibitor the during a 6 ns molecular dynamics simulation in the reconstituted (see Material and methods) co-crystalstructure of PKBa/Akt1 with Akt inhibitor VIII. In less than 500 ps the distance increase from 1 Å to more than 5 Å. (For interpretationof the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Role of PKBa/Akt1 Glu267-Lys268-Asn269 loop in Akt inhibitor VIII binding. A) Image of the co-crystal structure of the PH(pink) and kinase (green) domains of PKBa/Akt1 with Akt inhibitor VIII (yellow sticks). The Trp80 residue from the PH domain is inred. The motif Glu267-Lys268-Asn269 (blue) from the kinase domain is within binding distance of the inhibitor. B) Alignment of thesequence of PKBa/Akt1 and PKBg/Akt3 from the amino acid 262 to 273 of PKBa/Akt1. In this region the residues Glu267-Lys268-Asn269 of PKBa/Akt1, are replaced by Gly–Lys in PKBg/Akt3. C) NIH 3T3 cells expressing GFP-PKBa/Akt1 (AKT1) or a mutant inwhich the residues of the loop Glu267-Lys268-Asn269 were changed for Gly–Lys (mutant AKT1 EKN-GK) were treated for 30 minwith different concentrations of Akt inhibitor VIII as indicated prior to 5 min PDGF stimulation. Thr308 (left) and Ser473 (right)phosphorylations were detected by western blot using phospho-specific antibodies, and bands were quantified using the Odyssey/LI-COR fluorescence detection system. The data are presented as a percentage of Thr308 or Ser473 phosphorylations upon maximumPDGF stimulation (5 min) for each construct. An unpaired t-test shows a significant change in the phosphorylation of Thr308 in themutant compared to the wild type at 1 mM (p ¼ 0.04, n ¼ 3) and at 2.5 mM (p ¼ 0.02, n ¼ 3) and Ser473 in the mutant compared tothe wild type at 1 mM (p ¼ 0.03, n ¼ 3) and at 2.5 mM (p ¼ 0.02, n ¼ 3). D) The graph shows the distance between the Lys268 and Aktinhibitor VIII during a 12 ns molecular dynamics run in the reconstituted (see Material and methods) co-crystal structure of PKBa/Akt1 with the inhibitor. The distance is maintained very short during the whole run (around 3 Å). (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Role of PKBa/Akt1 Gln203 in Akt inhibitor VIII binding. A) Image of the dynamic model of the PH (pink) and kinase (green)domains of PKBa/Akt1 in the inactive PH-in conformation. The binding of the PH domain induces a cavity in the kinase domain.Through the cavity the PH domain residue Trp80 (red) becomes accessible from the outside. The image shows the docking of Aktinhibitor VIII (yellow sticks) inside the cavity. The imidazoquinoxaline moiety is at binding distance from Trp80 and the benzimi-dazolone moiety forms an H-bond with the residue Gln203 (purple). The Gln218 conserved in all PKB/Akt isoforms is in magenta. B)Comparison of the structure of Akt inhibitor VIII and the derived compound used in a clinical trial, MK-2206. In MK-2206 thebenzimidazolone moiety has been replaced with a four carbon cyclic moiety and an NH2. C) Alignment of PKBa/Akt1, PKBb/Akt2 andPKBg/Akt3 sequences from amino acid 199 to 215 of PKBa/Akt1. In this region the residue Gln203 of PKBa/Akt1, conserved in PKBb/Akt2 is a lysine in PKBg/Akt3. The replacement of Gln203 by a lysine in PKBa/Akt1 would induce the loss of the H-bond. D) NIH 3T3cells expressing GFP-PKBa/Akt1 (AKT1) or a mutant in which Gln203 was changed to a lysine (Q203K) were treated for 30 min withdifferent concentrations of AKT inhibitor VIII as indicated prior to 5 min PDGF stimulation. Thr308 (left) and Ser473 (right) phos-phorylations were detected by western blot using phospho-specific antibodies, bands were quantified using the Odyssey/LI-CORfluorescence detection system. The data are presented as a percentage of Thr308 or Ser473 phosphorylations upon maximumPDGF stimulation (5 min) for each construct. An unpaired t-test shows a significant change in the phosphorylation of Ser473 in themutant (AKT1-Q203K) compared to the wild type at 1.5 mM (p ¼ 0.008, n ¼ 6) and at 2.0 mM (p ¼ 0.001, n ¼ 6). (For interpretation ofthe references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Role of the PKBa/Akt1 C-terminal domain in Akt inhibitor VIII binding. A) Images of the dynamic model of the PH (pink) andkinase (green) domains of PKBa/Akt1 in the inactive PH-in conformation. The PH domain residue Trp80 is shown in red. Based onthe crystal structure of Barford and collaborators we performed the dynamic positioning of the C-terminal domain in the context ofboth PH and kinase domains of PKBa/Akt1 inactive conformation in panel a. Interestingly the Gln203 (purple) of the kinase domainis positioned at binding distance from the C-terminal regulatory domain (orange ribbon). The conserved Gln218 residue (magenta)that is critical for the positioning of the C-terminus is shown to be very close to Ser473 (cyan). The image in panel b shows thedocking of Akt inhibitor VIII (yellow sticks) inside of the cavity. The piperidinium moiety forms an H-bond with the residue Gln203and is very close to Ser473 of the C-terminal regulatory domain. The imidazoquinoxaline moiety is at binding distance of Trp80. C)Image of the co-crystal structure of PKBa/Akt1 PH and kinase domains with Akt inhibitor VIII (yellow sticks). Residues Gln203(purple) and Gln218 (magenta) are positioned far away from the inhibitor binding site. (For interpretation of the references to colourin this figure legend, the reader is referred to the web version of this article.)

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mutant could induce a rotation of the benzimidazolone moiety away from the Ser473 (Fig. 4A panel a)promoting a better accessibility to mTORC2. In addition it would also be in agreement with the fact thatthe position of the imidazoquinoxaline should not be affected by the mutation, observed by themaintenance of inhibition of Thr308. The crystal structure of the C-terminal domain bound to theisolated kinase domain of PKBb/Akt2 was published by Barford and collaborators (Yang et al., 2002a,b).Based on this crystal structure we performed the dynamic positioning of the C-terminal domain in thecontext of both PH and kinase domains of the PKBa/Akt1 inactive conformation that we published in anearlier study (Calleja et al., 2009). Fig. 4A panel b shows the position of the C-terminal domain (orangeribbon) in our dynamic model of PKBa/Akt1 PH-in conformation passing through the Trp80-inducedcavity. The C-terminus is located between the residues of the kinase domain Gln203 (dark purple)and Gln218 (magenta). Therefore our model suggests that the binding site for Akt inhibitor VIII wouldbe situated in the vicinity of the C-terminal domain close to the glutamines 203 and 218 and alsoSer473, in accordance with our biochemical data. However this does not seem to be the case in the co-crystal structure where the inhibitor buried at the interface between the PH and the kinase domainsand at a distance from the two glutamines.

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To summarize, the authors of the co-crystal structure of PKBa/Akt1 with Akt inhibitor VIII (Wu et al.,2010) identified two regions of PKBa/Akt1 within binding distance of the inhibitor that were differentin the three PKB/Akt isoforms. In particular these regions were different in PKBg/Akt3, which couldexplain its lower sensitivity. First, the Ser205 of PKBa/Akt1 was shown to be the only residue formingan H-bondwith Akt inhibitor VIII. However, its mutation to an alanine did not showa loss of PKBa/Akt1sensitivity, indicating that this residue was unlikely to have a role in the binding of the inhibitor in vivo.A significant decrease in the sensitivity to the inhibitor was seen in the case of the mutation of thesecond site the Glu267-Lys268-Asn269 loop, but only at suboptimal doses of inhibitor. These resultsindicated that the inhibitor binding required the cooperation of several weak interactions that mightnot all have been identified in the crystal structure.

Since the last 37 residues encompassing the C-terminal domain are lacking in the co-crystalstructure our intention was to investigate whether it might have a role in inhibitor binding. To do sowe used molecular modeling and dynamics simulations. In an earlier study we showed that the C-terminal domain of PKB/Akt, and in particular the residue Ser473 were in close proximity to Gln218(Fig. 4A panel b), a residue conserved in all three PKB/Akt isoforms. We proposed that this residue wasimportant for stabilization of the C-terminus in the kinase domain of PKB/Akt. Accordingly we showedthat the mutation of Gln218 residue to alanine induced the loss of Ser473 phosphorylation uponstimulation due to increased dephosphorylation (Calleja et al., 2009). Interestingly the mis-positioningof the C-terminal domain due to the Q218A mutation induced an increase in the sensitivity of PKBa/Akt1 to Akt inhibitor VIII at low doses of inhibitor (1 mM) suggesting that the binding site of theinhibitor was in close proximity or overlapping with the binding site of the C-terminal domain (Callejaet al., 2009). Furthermore the deletion of the last 39 amino acids of PKBa/Akt1, also induced an increasein the sensitivity to Akt inhibitor VIII (Calleja et al., 2009), reinforcing the notion that the C-terminaldomain and the inhibitor were bound in close proximity. In this study, from our docking model, weobserved that the carbonyl of the Gln203 could make an H-bond with the Hþ of the piperidiniummoiety of the inhibitor. The mutation of Gln203 for a lysine clearly showed a reduction of the Ser473phosphorylation inhibition. In agreement with our model the loss of the H-bond suggested a re-positioning of the benzimidazolone moiety of Akt inhibitor VIII away from Ser473 and indicatedfurther that the inhibitor would bind adjacent to the C-terminus. The inhibition of the Thr308 phos-phorylation, which is dependent on the locking of PKB/Akt in the PH-in conformation, was notmodified by the mutation. This would also be in accordance with the model since the mutation Q203Koutside of the cavity would not preclude the docking of the imidazoquinoxalinemoiety inside. It wouldbe interesting to study the effect of MK-2206 that is lacking the benzimidazolone on the Ser473sensitivity. Fig. 4A panel b shows the close proximity of the C-terminus to Gln218 and on the other sideto Gln203. Fig. 4A panel a shows the positioning of Akt inhibitor VIII derived from the moleculardocking (Fig. 3A) is in close proximity to both Gln218 and Gln 203. However, in the co-crystal structure(Fig. 4B) the inhibitor is shown buried inside of the PH and kinase domain interface far away from thetwo glutamines (Gln203 and Gln218). Unfortunately, as the C-terminus is missing from the crystalstructure it is difficult to speculate about its positioning. However it is clear that it could not be in theposition shown in the structure published by Barford and collaborators since it would clash withresidues of the kinase domain (not shown), though it is possible that binding of the inhibitor wouldinduce a change in the binding of the C-terminal domain in the kinase. However, our mutagenesisexperiments suggest that these four key features - Gln203 and Gln218, the C-terminus and Aktinhibitor VIII - would be in close proximity. It is therefore very difficult to reconcile the effect of ourmutations with the position of these four features observed in the co-crystal structure (Fig. 4B).

It is conceivable that the lack of a C-terminal regulatory domain in the co-crystal structuremay haveled to an alternative position of the inhibitor that might not be the one occurring in the full-lengthprotein. Alternatively we could imagine that depending on the activation status of PKB/Akt theinhibitor might target different binding sites. The position of the inhibitor in the co-crystal structureclearly indicates that a “breathing” of the protein would be absolutely necessary for the inhibitor toenter. This could happen in a situationwhere PKB/Akt would be in an active PH-out conformationwiththe PH and kinase domains separated. In an inactive PH-in conformation, binding might occur on thecavity formed by the PH and kinase domains, close to where the C-terminus is positioned. In this case,binding would not depend on a “breathing” of the protein. Further experiments will be needed to test

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our hypothesis. In particular the effect of MK-2206 might provide new insights into the mechanism ofthese allosteric inhibitors and additionally could lead to a more profound understanding of thecomplex mechanisms involved in PKBa/Akt1 regulation by inter-domain interactions.

Summary

Protein kinase B (PKB/Akt) is a serine/threonine kinase that plays a fundamental role in cell biologyand was found to be extremely deregulated in cancer. To prevent the aberrant activation of PKB/Akt intumors a series of highly specific allosteric inhibitors were developed (Akt inhibitor VIII also called dualAkt1/2 inhibitor). We have previously published a dynamic model of Akt inhibitor VIII binding withPKBa/Akt1 that explained the mechanism of inhibition due to the locking of the protein in its inactive(PH-in) conformation and also depicted the loss of PKBa/Akt1 Thr308 regulatory site phosphorylation.Recently, the co-crystal structure of PKBa/Akt1 with Akt inhibitor VIII confirmed this mechanism ofinhibition by locking components together. However the absence of the C-terminal domain in thecrystal structure did not allow the identification of the mechanism preventing the second regulatorysite (Ser473) phosphorylation that occurs upon inhibitor binding in vivo. In this study, based on ourdynamic model and the co-crystal structure, we identified and mutated residues predicted to have animpact on the sensitivity of PKBa/Akt1 and PKBg/Akt3 for Akt inhibitor VIII. Comparison of the datawith the predictions suggested that the C-terminal regulatory domain of PKB/Akt was likely to play animportant role in the positioning of Akt inhibitor VIII in vivo.

Acknowledgments

The authors would like to thank William Sellers for the construction of the Addgene plasmid 9017,and Nirmal Jethwa for critical reading of the manuscript.

Author contributions: VC was responsible for the design of the molecular biology tools and per-formed the biochemical experiments. ML was responsible for the molecular modeling and dynamics.VC, ML, and BL were involved in the project strategy and in scientific discussions. BL supervised theproject. VC, ML, and BL were involved in writing the manuscript.

The project was funded by the core funding of Cancer Research UK to Lincoln’s Inn Fields Labora-tories (London Research Institute- CR-UK-London).

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