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Interdomain Conformational Changes in Akt Activation Revealed by Chemical Cross-linking and Tandem Mass Spectrometry*

  • Bill X. Huang
    Affiliations
    From the Section of Mass Spectrometry, Laboratory of Membrane Biophysics and Biochemistry, NIAAA, National Institutes of Health, Bethesda, Maryland, 20892-9410
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  • Hee-Yong Kim
    Correspondence
    To whom correspondence should be addressed: Section of Mass Spectrometry, Laboratory of Membrane Biophysics and Biochemistry, NIAAA, National Institutes of Health, 5625 Fishers Lane, Bethesda, MD 20892-9410. Tel.: 301-402-8746; Fax: 301-594-0035;
    Affiliations
    From the Section of Mass Spectrometry, Laboratory of Membrane Biophysics and Biochemistry, NIAAA, National Institutes of Health, Bethesda, Maryland, 20892-9410
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      Akt, a serine/threonine kinase, plays a critical role in cell survival. Upon growth factor receptor stimulation, cytosolic Akt is recruited to the plasma membrane by phospholipid binding and activated through phosphorylation at Thr308 and Ser473. Although crystal structures for the parts of Akt have been reported, neither the three-dimensional structure of the whole molecule nor sequential conformational changes during activation have been demonstrated. In this study, we demonstrated that Akt undergoes dramatic interdomain conformational changes during activation processes by probing the three-dimensional structure of full-length Akt in solution using chemical cross-linking and tandem mass spectrometry. The cross-linking results not only provided new structural information but also revealed distinctive spatial arrangements of individual domains in the Akt molecule in resting, membrane-interacted, phosphorylated, and substrate-bound states. Our data allowed a new model for stepwise interdomain conformational changes in Akt activation sequence, setting a stage for the further investigation on Akt-membrane, Akt-protein, and/or Akt-drug interactions in solution to understand molecular mechanisms involved in physiological and pathophysiological processes of cell survival.
      The serine/threonine kinase Akt (protein kinase B (PKB)
      The abbreviations used are: PKB, protein kinase B; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PH, pleckstrin homology; 3D, three-dimensional; BS3, bis(sulfosuccinimidyl) suberate; DSS, disuccinimidyl suberate; DSG, disuccinimidyl glutarate, AMP-PNP, adenylyl imidodiphosphate; PS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; PC, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine; PE, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; GSK3, glycogen synthase kinase 3; PDK, phosphoinositide-dependent kinase.
      1The abbreviations used are: PKB, protein kinase B; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PH, pleckstrin homology; 3D, three-dimensional; BS3, bis(sulfosuccinimidyl) suberate; DSS, disuccinimidyl suberate; DSG, disuccinimidyl glutarate, AMP-PNP, adenylyl imidodiphosphate; PS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; PC, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine; PE, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; GSK3, glycogen synthase kinase 3; PDK, phosphoinositide-dependent kinase.
      ) is a central mediator for many cellular responses involved in cell survival, differentiation, proliferation, and insulin signaling (
      • Brazil D.P.
      • Hemmings B.A.
      Ten years of protein kinase B signalling: a hard Akt to follow.
      ,
      • Lawlor M.A.
      • Alessi D.R.
      PKB/Akt: a key mediator of cell proliferation, survival and insulin responses?.
      ). In human there are two major Akt isomers, Akt1 (PKBα) and Akt2 (PKBβ) (
      • Staal S.P.
      Molecular cloning of the Akt oncogene and its human homologues Akt1 and Akt2: amplification of Akt1 in a primary human gastric adenocarcinoma.
      ), which exhibit approximately 80% amino acid sequence identity. Each isomer possesses an N-terminal pleckstrin homology (PH) domain (residues 1–113), a kinase domain (residues 150–408) that is similar to those found in other AGC members such as cAMP-dependent protein kinase and protein kinase C, and a C-terminal regulatory domain (residues 409–480) containing a hydrophobic motif (Fig. 1) (
      • Bellacosa A.
      • Testa J.R.
      • Staal S.P.
      • Tsichlis P.N.
      A retroviral oncogene, Akt, encoding a serine-threonine kinase containing an SH2-like region.
      ,
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      Mechanism of activation and function of protein kinase B.
      ,
      • Thomas C.C.
      • Deak M.
      • Alessi D.R.
      • van Aalten D.M.F.
      High-resolution structure of the pleckstrin homology domain of protein kinase B/Akt bound to phosphatidylinositol (3,4,5)-trisphosphate.
      ). During the activation process, cytosolic Akt is recruited to the plasma membrane through the interaction of its PH domain with phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is generated by phosphatidylinositol 3-kinase in response to the growth factor receptor stimulation. It is thought that the membrane interaction results in conformational changes of Akt, exposing a pair of residues, Thr308 at the activation loop of the kinase domain and Ser473 at the hydrophobic motif of the regulatory domain, for phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) and a putative kinase PDK2, respectively (
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
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      Dual role of posphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B.
      ,
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      Role of translocation in the activation and function of protein kinase B.
      ,
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      • Ahmed N.N.
      • Datta K.
      • Malstrom S.
      • Stpkoe D.
      • McCormick F.
      • Feng J.N.
      • Tsichlis P.
      Akt activation by growth factors is a multiple-step process: the role of the PH domain.
      ,
      • Watton S.J.
      • Downward J.
      Akt/PKB localisation and 3′ phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction.
      ). It has been demonstrated that the phosphorylation at both Thr308 and Ser473 is required for the full activation of Akt (
      • Yang J.
      • Cron P.
      • Thompson V.
      • Good V.M.
      • Hess D.
      • Hemmings B.A.
      • Barford D.
      Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation.
      ). The phosphorylated active Akt in turn phosphorylates downstream effectors such as GSK3, PFK2, and BAD (
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      Cellular survival: a play in three Akts.
      ,
      • Datta S.R.
      • Dudek H.
      • Tao Masters, X.
      • Fu S.
      • Haian
      • Gotoh Y.
      • Greenberg M.E.
      Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.
      ).
      Figure thumbnail gr1
      Fig. 1Primary sequence of Akt1 showing three domains, the N-terminal PH domain (residues 1–113, marked in yellow), the kinase domain (residues 150–408, marked in red), and the C-terminal regulatory domain (residues 409–480, marked in green). Mass spectrometric detection of the tryptic digests covered 85% of the amino acid sequence (underlined) under our experimental conditions.
      Structural studies to understand the molecular mechanism for the activation and regulation of Akt have been carried out mostly using x-ray crystallography. To date, high resolution crystal structures of Akt have been reported for the Akt1 PH domain with and without PIP3 binding (
      • Thomas C.C.
      • Deak M.
      • Alessi D.R.
      • van Aalten D.M.F.
      High-resolution structure of the pleckstrin homology domain of protein kinase B/Akt bound to phosphatidylinositol (3,4,5)-trisphosphate.
      ,
      • Milburn C.C.
      • Deak M.
      • Kelly S.M.
      • Price S.M.
      • Alessi D.R.
      • van Aalten D.M.F.
      Binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change.
      ), the unliganded inactive Akt2 kinase domain (
      • Huang X.
      • Begley M.
      • Morgenstern K.A.
      • Gu Y.
      • Rose P.
      • Zhao H.
      • Zhu X.
      Crystal structure of an inactive Akt2 kinase domain.
      ), and the kinase domain of active Akt2 complexed with GSK3β peptide and an ATP analog (
      • Yang J.
      • Cron P.
      • Good V.M.
      • Thompson V.
      • Hemmings B.A.
      • Barford D.
      Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP.
      ). Studies using NMR or circular dichroism have also provided supportive information on the conformation of Akt bound to PIP3 (
      • Milburn C.C.
      • Deak M.
      • Kelly S.M.
      • Price S.M.
      • Alessi D.R.
      • van Aalten D.M.F.
      Binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change.
      ,
      • Auguin D.
      • Barthe P.
      • Auge-Senegas M.
      • Stern M.
      • Noguchi M.
      • Roumestand C.
      Solution structure and backbone dynamics of the pleckstrin homology domain of the human protein kinase B (PKB/Akt): interaction with inositol phosphates.
      ). In addition, fluorescence lifetime imaging microscopy has been used to monitor in situ conformational changes in response to growth factor stimulation (
      • Calleja V.
      • Ameer-beg S.M.
      • Vojnovic B.
      • Woscholski R.
      • Downward J.
      • Larijani B.
      Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy.
      ). Nevertheless up to now these methods demonstrated neither the 3D structure of the whole Akt molecule nor sequential conformational changes involved in the activation process. Apparently an alternative technique to explore the 3D structures of the full-length Akt is required for further understanding of this enzyme. Chemical cross-linking combined with MS has recently emerged as a sensitive tool for probing 3D structure of proteins in solution (
      • Young M.M.
      • Tang N.
      • Hempel J.C.
      • Oshiro C.M.
      • Taylor E.W.
      • Kuntz I.D.
      • Gilson B.W.
      • Dollinger G.
      High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry.
      ,
      • Taverner T.
      • Hall N.E.
      • Ohair R.A.J.
      • Simpson R.J.
      Characterization of an antagonist interleukin-6 dimer by stable isotope labeling, cross-linking, and mass spectrometry.
      ,
      • Back J.W.
      • de Jong L.
      • Muijsers A.O.
      • Koster C.G.
      Chemical cross-linking and mass spectrometry for protein structural modeling.
      ,
      • Huang B.X.
      • Dass C.
      • Kim H.-Y.
      Probing conformational changes of human serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry.
      ,
      • Silva R.A.G.
      • Hilliard G.M.
      • Fang J.
      • Macha S.
      • Davidson W.S.
      A three-dimensional molecular model of lipid-free apolipoprotein A-I determined by cross-linking/mass spectrometry and sequence threading.
      ). Determination of the cross-linked amino acid residues by MS provides spatial distance information that has been proven valuable for the elucidation of protein structural changes due to protein-ligand and protein-protein interactions in more physiologically relevant conditions (
      • Bennett K.L.
      • Kussmann M.
      • Bjork P.
      • Godzwon M.
      • Mikkelsen M.
      • Sorensen P.
      • Roepstorff P.
      Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping—a novel approach to assess intermolecular protein contacts.
      ,
      • Sinz A.
      Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes.
      ,
      • Zhang X.
      • Wehbi H.
      • Roberts M.F.
      Cross-linking phosphatidylinositol-specific phospholipase C traps two activating phosphatidylcholine molecules on the enzyme.
      ,
      • Giron-Monzon L.
      • Manelyte L.
      • Ahrends R.
      • Kirsch D.
      • Spengler B.
      • Friedhoff P.
      Mapping protein-protein interactions between MutL and MutH by cross-linking.
      ,
      • Chu F.
      • Shan S.
      • Moustakas D.T.
      • Alber F.
      • Egea P.F.
      • Stroud R.M.
      • Walter P.
      • Burlingame A.L.
      Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry.
      ,
      • Huang B.X.
      • Dass C.
      • Kim H.-Y.
      Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry.
      ). This approach complements, and sometimes is a viable alternative to, x-ray crystallography, particularly when a whole protein molecule cannot be readily crystallized.
      In the present study, we probed the 3D structures of full-length Akt1 (Akt hereafter) in solution using lysine-specific bifunctional cross-linkers with varying spacer arm lengths and chemistry, including bis(sulfosuccinimidyl) suberate (BS3), disuccinimidyl suberate (DSS), and disuccinimidyl glutarate (DSG), to provide spatial distance constraints between Akt domains yet to be established. To gain insights into the Akt activation mechanism, Akt conformation at different activation stages, including inactive, membrane-interacted, phosphorylated, and substrate-bound states was probed. The cross-linked lysine residues were unambiguously determined by tandem mass spectrometry with static nano-ESI and HPLC/nano-ESI. 18O labeling of tryptic digests (
      • Yao X.
      • Freas A.
      • Ramirez J.
      • Demirev P.A.
      • Fenselau C.
      Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus.
      ) was used to further confirm the through-space cross-linked peptides (
      • Huang B.X.
      • Dass C.
      • Kim H.-Y.
      Probing conformational changes of human serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry.
      ,
      • Huang B.X.
      • Dass C.
      • Kim H.-Y.
      Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry.
      ,
      • Back J.W.
      • Notenboom V.
      • Koning L.J.
      • Muijsers A.O.
      • Sixma T.K.
      • Koster C.G.
      • Jong L.
      Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling.
      ) and to compare the peak intensity of the cross-linked peptides produced from Akt at various stages of activation. We identified two interdomain cross-linked pairs that allowed us to monitor interdomain conformational changes during activation. These cross-linked pairs also provided spatial distance constraints between domains that are not available from the crystal structure. Our results demonstrate for the first time that Akt undergoes distinctive interdomain conformational changes at each step of activation and substrate binding.

      EXPERIMENTAL PROCEDURES

       Chemicals—

      Inactive and phosphorylated active Akt1 with 90% purity was purchased from Upstate Cell Signaling Solutions (Lake Placid, NY) or Calbiochem. BS3, DSS, and DSG were purchased from Pierce. Modified trypsin was purchased from Promega (Madison, WI). K-LISA Akt activity kit and adenylyl imidodiphosphate (AMP-PNP) tetralithium salt were purchased from Calbiochem. Phospho-Thr308 and -Ser473 Akt antibodies were purchased from Cell Signaling Technology (Beverly, MA). GSK3β peptide substrate (GRPRTTSFAE) was purchased from AnaSpec Inc. (San Jose, CA). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (PS), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PC), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE) were purchased from Avanti Polar Lipids (Alabaster, AL). l-α-d-myo-Phosphatidylinositol 3,4,5-trisphosphate was purchased from A. G. Scientific, Inc. (San Diego, CA). HEPES buffer solution was purchased from Invitrogen. Pure water was obtained from a Gemini high purity water system (West Berlin, NJ). H218O (99%) was purchased from Isotec (Miamisburg, OH). Dimethyl sulfoxide (Me2SO) was purchased from Sigma. Other chemicals were purchased from Mallinckrodt.

       Cross-linking Reaction and Tryptic Digestion—

      Akt samples at 5 μm were dialyzed overnight against 50 mm HEPES (pH 7.8) containing 50 mm NaCl at 4 °C to remove the primary amine-containing Tris-HCl buffer. Aliquots before or after dialysis were used to measure the Akt activity and subjected to Western blotting against phosphospecific Akt antibodies. The Akt activity measured before and after dialysis was identical, although the protein lost ∼30% of its activity under the conditions used in this study. The samples were incubated with a 50 molar excess of freshly prepared BS3 (in pH 5.0 sodium citrate), DSS, or DSG (in Me2SO at a final concentration of 1% in the reaction mixture) at room temperature for 5 min. At this cross-linking condition intermolecular cross-linked dimers or multimers were not observed according to the SDS-PAGE separation data. The cross-linking reaction was quenched with 1 m Tris-HCl (pH 7.4). The samples were digested with trypsin at 37 °C for 20 h using a trypsin to protein ratio of 1:20. Alternatively samples were reduced with dithiothreitol and alkylated with iodoacetamide before tryptic digestion. For 18O labeling of tryptic digests, samples were dialyzed against water, dried using a vacuum centrifuge, and reconstituted in pure water or 99% H218O with 5% acetonitrile prior to the digestion with trypsin (
      • Huang B.X.
      • Dass C.
      • Kim H.-Y.
      Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry.
      ,
      • Back J.W.
      • Notenboom V.
      • Koning L.J.
      • Muijsers A.O.
      • Sixma T.K.
      • Koster C.G.
      • Jong L.
      Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling.
      ).

       Akt Kinase Assay and Western Blot—

      The kinase activity was measured with a K-LISA Akt activity kit that uses streptavidin-coated 96-well plates and a biotinylated peptide substrate (GRPRTSSFAEG) according to the manufacturer’s instructions (Calbiochem). The phosphorylation states of Akt were examined by Western blots using phospho-Thr308 and -Ser473 Akt antibodies.

       Preparation of Unilamellar Vesicles and Akt-Liposome Interaction—

      Unilamellar vesicles containing PE, PC, PS, and PIP3 at a ratio of 50:18:30:2 were prepared according to the method reported previously (
      • Kim H.-Y
      • Bigelow J.
      • Kevala J.H.
      Substrate preference in phosphatidylserine biosynthesis for docosahexaenoic acid containing species.
      ). After dialysis overnight against 50 mm HEPES (pH 7.8) containing 50 mm NaCl at 4 °C, inactive Akt was incubated with the freshly prepared unilamellar vesicles at 37 °C for 1 h (
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.J.
      • Reese C.B.
      • Cohen P.
      Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates protein kinase Bα.
      ).

       Complex Formation with GSK3β Peptide and AMP-PNP—

      Active or inactive Akt at 5 μm was incubated with equivalent volumes of 50 μm GSK3β peptide substrate and 200 μm AMA-PNP (
      • Yang J.
      • Cron P.
      • Good V.M.
      • Thompson V.
      • Hemmings B.A.
      • Barford D.
      Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP.
      ) at room temperature for 1 h and dialyzed overnight against 50 mm HEPES (pH 7.8) containing 50 mm NaCl at 4 °C. After treatment with cross-linkers, aliquots were subjected to tryptic digestion followed by mass spectrometric analysis in accordance with the procedures described under “Cross-linking Reaction and Tryptic Digestion.”

       Off-line Static Nano-ESI Mass Spectrometric Analysis—

      Tryptic digests were desalted with a C18 ZipTip (Millipore Corp.) prior to analysis by a high resolution QSTAR pulsar Qq-TOF mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, CA) equipped with a nanoelectrospray ionization source. The ion source voltage was set at 1100 V in the positive ion mode. A full mass spectrum was acquired over an m/z range of 500–2000, and the ions of interest were subjected to CID using high purity argon to obtain tandem MS data. The performance of the instrument featured resolution greater than 8000 and mass accuracy with less than 30 ppm error in both full MS and tandem MS mode. The m/z data were reconstructed to generate mass spectra using Analyst QS software (Applied Biosystems/MDS Sciex).

       HPLC/Nano-ESI Mass Spectrometric Analysis—

      HPLC/nano-ESI MS analysis was performed on an Agilent ion trap mass spectrometer (XCT) equipped with an Agilent 1100 nanoflow HPLC system. Peptides were trapped in a C18 enrichment column (Zorbax 300SB C18, 0.3 × 5 mm, Agilent, Wilmington, DE) and then separated on a Zorbax 300SB C18 column (75 μm × 150 mm, 3.5 μm) using a mobile phase that contained solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min. The mobile phase composition was held initially at 3% B for 5 min and gradually changed to 15, 45, and 85% B at 8, 50, 55 min, respectively, and held at 85% for 5 min. The data-dependent automatic scan mode (“Auto-MS2”) was used. The capillary voltage was set to 1750 V. The mass spectrometer was scanned from 300 to 2000 m/z range.

       Analysis of the Cross-linked Peptides—

      Protein Analysis Work Sheet (PAWS) was used to assign the mass values of tryptic peptides. The emerging cross-linked peptides were identified by comparing the mass spectra of tryptic peptides obtained from modified sample with those from the unmodified control. Tandem MS data for the cross-linked peptides were manually interpreted with the assistance of Protein Analysis Work Sheet and Analyst QS software (Applied Biosystem/MDS Sciex).

      RESULTS AND DISCUSSION

       Determination of the Cross-linked Residues in Inactive Akt—

      The tryptic peptides of Akt detected by nano-ESI MS represented more than 85% of sequence coverage (Fig. 1). The cross-linked peptide peaks were distinguished by comparing the static nano-ESI mass spectra obtained from cross-linker-modified Akt with the mass spectrum from the non-modified control. Fig. 2 shows reconstructed mass spectra of the tryptic digests from the control and modified Akt. Twelve newly emerging peaks were identified in Akt samples modified with DSS. Modification with BS3, a hydrophilic analog of DSS, yielded the same results, indicating that all the modified lysine residues are exposed on the surface and easily accessible by both hydrophilic and hydrophobic cross-linking reagents. DSG, which contains a cross-linking arm (C5H6O2) shorter by (CH2)3 than that of DSS (C8H12O2), was used to refine distance constraints for the observed lysine pairs. In the DSG-modified sample, all but one of the newly emerging peaks were observed at mass values 42 Da lower than those observed in the DSS-modified sample. The through-space cross-linked peptide peaks, which are more valuable with respect to protein folding, were further confirmed by 18O labeling of the tryptic digests. When hydrolyzed in H218O, each C terminus of a tryptic peptide incorporates two 18O atoms (
      • Yao X.
      • Freas A.
      • Ramirez J.
      • Demirev P.A.
      • Fenselau C.
      Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus.
      ). Thus, internally cross-linked or end-capped peptides show a 4-Da mass shift, whereas the through-space cross-linking between two peptides results in an 8-Da mass increase (
      • Huang B.X.
      • Dass C.
      • Kim H.-Y.
      Probing conformational changes of human serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry.
      ,
      • Huang B.X.
      • Dass C.
      • Kim H.-Y.
      Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry.
      ,
      • Back J.W.
      • Notenboom V.
      • Koning L.J.
      • Muijsers A.O.
      • Sixma T.K.
      • Koster C.G.
      • Jong L.
      Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling.
      ). The peptides with masses of 2458, 3465, and 3623 Da, reconstructed from the corresponding multiply charged ions, increased 8 Da in mass when labeled with 18O, indicating that these peptides resulted from through-space cross-linking. The rest of the emerging peptides showed 4-Da mass shifts indicating that they originated from the end capping or internal cross-linking.
      Figure thumbnail gr2
      Fig. 2Nano-ESI-Qq-TOF reconstructed mass spectra of tryptic digests from unmodified or cross-linker-modified inactive Akt samples. Newly emerging cross-linked peptides (marked with asterisks) were identified in modified samples. 18O labeling facilitated the identification of three through-space cross-linked peptides by showing an 8-Da mass shift as demonstrated by DSS-modified samples (mass shifted pairs are shown in bold).
      Among the three through-space cross-linked pairs observed in DSS- and BS3-modified Akt, the peaks with 2458 and 3465 Da turned out to be interdomain cross-linked peptide pairs according to the MS/MS analysis (Fig. 3). Fig. 3 (top) shows the MS/MS spectrum from the peptide with mass of 2458 Da (reconstructed from the triply charged ion at m/z 820.4) observed in the DSS- and BS3-modified samples. The spectrum revealed that this peptide resulted from the cross-linking between the segments Tyr26–Lys39 and Asp387–Arg391 with Lys30 of the PH domain linked to Lys389 of the kinase domain via C8H10O2 (derived from C8H12O2, the cross-linking arm of DSS, with two hydrogen atoms subtracted due to cross-linking). The Lys30-Lys389 cross-linking observed in the DSS-modified sample (2458 Da) was not detected from the DSG-modified Akt (supposedly 2416 Da). The sequence assignment was based on the fragment ions observed for both segments and the ions containing C8H10O2. The N-terminal “b” or “a” ions and C-terminal “y” ions from each segment were separately labeled using subscripts α and β (
      • Schilling B.
      • Row R.H.
      • Gilbon B.W.
      • Guo X.
      • Yong M.M.
      MS2assign, automated assignment and nomenclature of tandem mass spectrometry of chemically cross-linked peptides.
      ). The MS/MS analysis also revealed the other interdomain cross-linking between Lys284 of the kinase domain and Lys426 of the regulatory domain that was detected at 3465 and 3423 Da after modification with DSS (or BS3) and DSG, respectively (Fig. 2). The MS/MS spectrum indicated that the peak at 3423 Da (reconstructed from the quadruply charged ion at m/z 856.7) consisted of the peptide segments Leu277–Lys289 and Leu421–Arg436 cross-linked between Lys284 and Lys426 via C5H4O2 (Fig. 3, bottom). Consistently the MS/MS spectra of the corresponding cross-linked peptide detected with mass of 3465 Da in the DSS- or BS3-modified samples revealed the same cross-linking with a longer bridge of C8H10O2 (data not shown).
      Figure thumbnail gr3
      Fig. 3MS/MS analysis of the cross-linked peptides with mass of 2458 Da (top) and 3423 Da (bottom) depicted in . The mass values of the peptides were derived from the corresponding multiply charged ions (insets). The sequences of the peptides were assigned with single letter abbreviation based on the fragment ions observed for the cross-linked peptide segments. N-terminal b ions and C-terminal y ions resulting from the amide bond cleavage as well as N-terminal a ions due to the cleavage of C–C bond are labeled. Top, the MS/MS spectrum indicated that two peptide segments, Tyr26–Lys39 (labeled with α) and Asp387–Arg391 (labeled with β), were cross-linked at Lys30 and Lys389 via C8H10O2. Bottom, the MS/MS spectrum indicated that Lys284 of the peptide segment Leu277–Lys289 (labeled with β) linked to Lys426 of Leu421–Arg436 (labeled with α) via C5H4O2, the cross-linking bridge from DSG.

       Probing 3D Structure of Inactive Akt Using Cross-linking Data—

      Table I summarizes all the cross-linked peptides identified and characterized by tandem MS, including two interdomain and five intradomain cross-linked pairs as well as five end-capped peptides. The presence of all these modified peptide peaks identified by static nano-ESI MS was also unambiguously confirmed by HPLC/MS/MS (data not shown). The α-carbon distance constraints for each cross-linked lysine pair determined in the present study are shown in Table I. Table I also lists the α-carbon distances between the corresponding lysine residues from the crystal structures of the inactive Akt1 PH domain (Protein Data Bank entry 1unp) or the kinase domain of inactive Akt2 (Protein Data Bank entry 1mrv) as the reference.
      Table ICross-linked lysine residues in inactive Akt molecule
      Cross-linked lysine pairs or end-capped lysine residues identified by MS/MSMass of the cross-linked tryptic peptidesType of cross-linkingBS3 -mod.DSS-mod.DSG-mod.Distance constraint of cross-linked α-carbonsα-Carbon distance in the crystal structure
      Da
      Lys111(PH)-Lys112(PH)3120
      Using DSS or BS3.
      (3078)
      Using DSG.
      Intradomain+++≤203.8
      Obtained from the Protein Data Bank entry 1unp.
      Lys214(kin)-Lys284(kin)3623 (3581)Intradomain+++≤2017.9
      Obtained from the equivalent lysine residues in inactive Akt2 (Protein Data Bank entry 1mrv).
      Lys158(kin)-Lys163(kin)1610 (1568)Intradomain+++≤204.0
      Obtained from the equivalent lysine residues in inactive Akt2 (Protein Data Bank entry 1mrv).
      Lys30(PH)-Lys39(PH)2939 (2897)Intradomain+++≤2017.9
      Obtained from the Protein Data Bank entry 1unp.
      Lys284(kin)-Lys426(reg)3465 (3423)Interdomain+++≤207.8
      Obtained from the equivalent lysine residues in inactive Akt2 (Protein Data Bank entry 1mrv).
      Lys30(PH)-Lys389(kin)2458 (ND)Interdomain++20–24NA
      Lys377(kin)-Lys385(kin)1792 (1750)Intradomain+++≤2012.1
      Obtained from the equivalent lysine residues in inactive Akt2 (Protein Data Bank entry 1mrv).
      Lys20(PH)1617 (1575)Capping+++
      Lys189(kin)2079 (2037)Capping+++
      Lys268(kin)2728 (2686)Capping+++
      Lys400(kin)1783 (1741)Capping+++
      Lys420(reg)2086 (2044)Capping+++
      a Using DSS or BS3.
      b Using DSG.
      c Obtained from the Protein Data Bank entry 1unp.
      d Obtained from the equivalent lysine residues in inactive Akt2 (Protein Data Bank entry 1mrv).
      Our results indicated that all the cross-linked or end-capped lysine residues were exposed on the surface because they were modified by both hydrophobic DSS and its hydrophilic analog BS3. The residues Lys189 and Lys268, which were missing in the crystal structure due to inadequate resolution of the electron density (
      • Huang X.
      • Begley M.
      • Morgenstern K.A.
      • Gu Y.
      • Rose P.
      • Zhao H.
      • Zhu X.
      Crystal structure of an inactive Akt2 kinase domain.
      ) or absence of the equivalent lysine residue, respectively, were also end-capped, revealing that these residues are in the easily accessible environment. The spatial distance constraint generated by DSS (or BS3) or DSG for cross-linked lysine pairs is 24 and 20 Å, respectively, considering the mobility of the side chains of lysine residues (
      • Young M.M.
      • Tang N.
      • Hempel J.C.
      • Oshiro C.M.
      • Taylor E.W.
      • Kuntz I.D.
      • Gilson B.W.
      • Dollinger G.
      High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry.
      ,
      • Huang B.X.
      • Dass C.
      • Kim H.-Y.
      Probing conformational changes of human serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry.
      ). The cross-linking was observed for Lys30-Lys39, Lys111-Lys112, Lys158-Lys163, Lys214-Lys284, Lys284-Lys426, and Lys377-Lys385 not only with DSS and BS3 but also with DSG, a shorter cross-linker, indicating that the distance between the α-carbons of the lysine residues in each pair is less than 20 Å. This result was in agreement with the x-ray crystallographic data reported for the PH domain of Akt1 and the kinase domain of Akt2. An exception was observed for the interdomain cross-linking between Lys30 of the PH domain and Lys389 of the kinase domain that was detected only with the longer cross-linkers DSS and BS3 but not in the DSG-modified sample, suggesting that the distance between the α-carbons of this lysine pair is greater than 20 Å but less than 24 Å. This interdomain distance constraint, which is not yet available from the existing x-ray crystallographic data, should be valuable for modeling a full-length Akt molecule. The presence of the two interdomain cross-linked pairs Lys30-Lys389 and Lys284-Lys426 indicated a spatial proximity between the PH and the kinase domains as well as the regulatory and kinase domains in the Akt structure, suggesting that inactive Akt exists as a folded molecule with the PH and regulatory domains covering parts of the kinase domain.

       Conformational Changes of Akt upon Membrane Interaction, Phosphorylation, and Substrate Binding—

      The two interdomain cross-linked pairs Lys30-Lys389 and Lys284-Lys426 allowed us to monitor Akt conformation at different stages of activation, including inactive, membrane-interacted, phosphorylated, and substrate-bound states. The activity and phosphorylation states of active Akt were evaluated by K-LISA activity assay, Western blotting, and mass spectrometry (Fig. 4). Only the active Akt showed kinase activity and phosphorylation at both Thr308 and Ser473 determined by the K-LISA assay and Western blotting described under “Experimental Procedures.” Consistently the tryptic peptides containing phosphorylated Thr308 (3362 Da) and Ser473 (1732 Da) were detected only in the mass spectrum of the active Akt sample. In comparison with the inactive Akt, non-phosphorylated tryptic peptides containing either Thr308 or Ser473 decreased by ∼80% in the active sample according to the quantitation based on 18O labeling and/or the intensity comparison using a known unmodified tryptic peptide (data not shown).
      Figure thumbnail gr4
      Fig. 4Evaluation of the kinase activity by K-LISA activity assay and the phosphorylation states by Western blotting (top) and mass spectrometry (bottom). Tryptic peptides containing phosphorylated Thr308 (3362 Da) and Ser473 (1732 Da) were detected only in the mass spectrum of the active Akt samples. pS, phosphoserine; pT, phosphothreonine.
      To observe the conformational changes upon membrane interaction, inactive Akt was incubated with liposomes containing PE/PC/PS/PIP3 at a ratio of 50:18:30:2, which approximated a lipid composition in the inner leaflet of the neuronal plasma membrane under a stimulated condition, considering the fact that PS is exclusively localized in the inner plasma membrane, whereas PC is mostly on the outside. Under this condition, the two interdomain cross-linked pairs Lys30-Lys389 and Lys284-Lys426 were no longer observed within 24-Å spatial distance constraint (Fig. 5, top). Because individual modification of these lysine residues was observed under this condition (data not shown), it is clear that these residues were still accessible, suggesting that the cross-linking partners in Lys30-Lys389 and Lys284-Lys426 moved further away from each other upon membrane interaction. In the phosphorylated active form of Akt, Lys284-Lys426 cross-linking was still missing, but Lys30-Lys389 cross-linking reappeared, indicating that the proximity between Lys30 and Lys389 was regained due to phosphorylation. When cross-linked with DSG, Lys30-Lys389 (supposedly with mass of 2416 Da) was not observed in the active form (Fig. 5, bottom), indicating that the spatial distance between Lys30 and Lys389 remained 20–24 Å as was the case with the inactive Akt.
      Figure thumbnail gr5
      Fig. 5Nano-ESI-Qq-TOF reconstructed mass spectra from modified Akt samples at different activation stages.Top, in DSS-modified samples, the peaks with masses of 2458 and 3465 Da representing the interdomain cross-linked pairs Lys30-Lys389 and Lys284-Lys426, respectively, were detected depending on the activation state. Bottom, in DSG-modified samples, the peak representing the cross-linked pair Lys284-Lys426 was observed in inactive Akt but not in active Akt, whereas Lys30-Lys389 (supposedly with mass of 2416 Da) was not observed in both cases. Cross-linked peptides are marked with asterisks.
      An analytical concern regarding the conclusion drawn from the comparison of peak abundance between non-identical complex mixtures is that peaks of interest may be reduced or eliminated due to ion suppression in different samples. To undoubtedly confirm the observed changes of the peaks, we took advantage of the 18O labeling technique. The cross-linker-modified inactive Akt sample digested in H218O was mixed with the modified Akt samples from different activation stages digested in normal H216O water, and the mixture was subjected to MS analysis. As an example, the mass spectrum is shown in Fig. 6 for the DSS-modified 18O-labeled digest of the inactive Akt mixed with the 16O digest of the DSS-modified active Akt at a ratio of 1:1. As expected the peak intensity ratio of the non-cross-linked tryptic peptide with mass of 2886 Da (Thr87–Lys111) in H216O digest to its counterpart of 2890 Da in H218O digest was ∼1:1. A similar ratio was observed for the cross-linked pair of Lys30-Lys389 (2458 versus 2466 Da) or the peak end-capped at Lys268 (2728 versus 2732 Da), indicating the presence of these cross-linked pairs in both inactive and active forms at a comparable level. However, Lys284-Lys426 cross-linking was detected only with the inactive Akt at 3473 Da (18O digest) and was not detected at 3465 Da with the active sample digested in H216O with the instrumental sensitivity of our current setting. These results were further confirmed by LC/MS/MS (data not shown).
      Figure thumbnail gr6
      Fig. 6Representative nano-ESI-Qq-TOF reconstructed mass spectrum obtained from the DSS-modified 18O-labeled tryptic digests of the inactive Akt mixed with the 16O digests of the active Akt at a ratio of 1:1. The zoomed-in views of the spectra are also shown. The peak with mass of 3465 Da representing the cross-linking of Lys284-Lys426 from active Akt (16O digests) was not detected in the spectrum. Other peaks including non-cross-linked peptide Thr87–Lys111 (T[87–111]K), end-capped peptide Phe252–Arg273 (F[252–273]R) with Lys268 capping, and Lys30-Lys389 were detected with intensities comparable to those from inactive Akt (18O digests).
      Conformational changes of Akt also occurred when active Akt was bound to substrates. The substrate binding status was indicated in the mass spectra obtained from the tryptic digest of the active Akt after the incubation with substrates. Binding the GSK3β peptide substrate and AMP-PNP, an ATP analog, to Akt interfered with the tryptic digestion at Lys307, resulting in the miscleavage at Lys307. A new peak with mass of 3965 Da was identified by MS/MS as Asp302–Arg328 and Lys290–Ile297 linked via a disulfide bond between Cys310 and Cys296 with phosphorylation at Thr308 (data not shown). This peak was not observed in the tryptic digest either from active Akt alone (phosphorylation at Thr308 alone did not interfere the tryptic hydrolysis at Lys307) or from inactive Akt mixed with GSK3β peptide and AMP-PNP (as the corresponding unphosphorylated peak at 3885 Da), indicating that the substrate was indeed bound to the kinase loop (residues 296–311) region specifically in active Akt. In the substrate-bound form, Lys284-Lys426 cross-linking (3465 Da), which was not observed in the unliganded active Akt form, emerged again with DSS (Fig. 5, top) as well as BS3 and DSG (data not shown).
      The intra-PH domain and intra-kinase domain cross-linking, including Lys30(β2)-Lys39(β3/β4 loop), Lys111(α-helix)-Lys112(α-helix), Lys158(β1)-Lys163(β2), Lys214(β4)-Lys284(β7/β8 loop), and Lys377(αH)-Lys385(αH/αI loop), remained unaltered after membrane interaction, phosphorylation, or substrate binding. Although the local conformational changes within each domain could not be probed by this approach due to the low resolution in probing distance constraints using current cross-linkers, the changes in the interdomain cross-linking observed in this study clearly indicated that arrangements of individual domains were profoundly affected during activation processes.

       Proposed Scheme for Interdomain Conformational Changes in Akt Activation—

      From the currently available biochemical data (
      • Brazil D.P.
      • Hemmings B.A.
      Ten years of protein kinase B signalling: a hard Akt to follow.
      ,
      • Alessi D.R.
      • Cohen P.
      Mechanism of activation and function of protein kinase B.
      ) it has been proposed that PIP3 interacts with the PH domain of Akt, exposing Thr308 and Ser473, which in turn are subjected to phosphorylation by upstream kinases for activation (
      • Brazil D.P.
      • Hemmings B.A.
      Ten years of protein kinase B signalling: a hard Akt to follow.
      ,
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.J.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      Dual role of posphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B.
      ). For example, mutating the PH domain abolishes the phosphorylation and activation of Akt by the upstream kinases even in the presence of PIP3 (
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.J.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      Dual role of posphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B.
      ). On the other hand, truncation of the PH domain results in phosphorylation and activation of Akt without PIP3 (
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.J.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      Dual role of posphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B.
      ). These data suggest that the PH domain may block the access of the upstream kinase to the phosphorylation sites of Akt, and the binding of PIP3 to the PH domain is a way to remove such structural hindrance. Additionally an in situ study using fluorescence lifetime imaging microscopy has suggested that Akt undergoes a conformational change at the plasma membrane upon growth factor stimulation in intact cells (
      • Calleja V.
      • Ameer-beg S.M.
      • Vojnovic B.
      • Woscholski R.
      • Downward J.
      • Larijani B.
      Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy.
      ). Nevertheless no structural evidence is available concerning the alteration of interdomain arrangements accompanying the activation sequence. The lack of the cross-linking of Lys30-Lys389 and Lys284-Lys426 in the liposome-interacted Akt molecule observed in our study provided evidence that Akt indeed exists with an open conformation upon phospholipid binding, presumably exposing the phosphorylation sites. Our data also revealed that this membrane-induced open conformation was subsequently altered by phosphorylation. The presence of Lys30-Lys389 in both inactive and phosphorylated forms suggests that the spatial arrangements of the PH and kinase domains are similar in both active and inactive forms. The fact that this cross-linking first disappeared due to membrane interaction before it was detected again in the phosphorylated form strongly suggests that phosphorylation triggered the separation of the PH domain from the membrane. The absence of the cross-linked pair Lys284-Lys426 within the distance constraint of 24 Å in both membrane-interacted and activated forms indicates that Lys284 and Lys426 moved farther away from each other during activation, at least by 16 Å, considering the reported distance between the corresponding residues (7.8 Å) from the crystal structure of inactive Akt2 (Table I). The crystal structure has revealed that the binding pockets for the substrate peptide and ATP, located within the activation loop and at the interface between the N-lobe (containing β1–5, αB, and αC) and C-lobe (mainly an α-helix), respectively, are blocked in the inactive Akt2 molecule (
      • Huang X.
      • Begley M.
      • Morgenstern K.A.
      • Gu Y.
      • Rose P.
      • Zhao H.
      • Zhu X.
      Crystal structure of an inactive Akt2 kinase domain.
      ,
      • Yang J.
      • Cron P.
      • Good V.M.
      • Thompson V.
      • Hemmings B.A.
      • Barford D.
      Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP.
      ) with the C-terminal chain covering part of the ATP binding site. In addition, it has been suggested that residues such as Phe439 of the regulatory domain assist stabilization of the blocking conformation (
      • Huang X.
      • Begley M.
      • Morgenstern K.A.
      • Gu Y.
      • Rose P.
      • Zhao H.
      • Zhu X.
      Crystal structure of an inactive Akt2 kinase domain.
      ), and a dramatic conformational change to unblock these binding sites is required for Akt activation (
      • Huang X.
      • Begley M.
      • Morgenstern K.A.
      • Gu Y.
      • Rose P.
      • Zhao H.
      • Zhu X.
      Crystal structure of an inactive Akt2 kinase domain.
      ,
      • Yang J.
      • Cron P.
      • Good V.M.
      • Thompson V.
      • Hemmings B.A.
      • Barford D.
      Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP.
      ). According to our data such dramatic displacement of the regulatory domain was introduced by the interaction with membrane, uncovering the binding pockets, and this open conformation was sustained after phosphorylation. Most probably, this open conformation allows substrate entry to the kinase domain, serving as an important determinant for the activated state of Akt in cytosol. The presence of cross-linked pair Lys284-Lys426 after binding to GSK3β peptide and AMP-PNP suggests that a closed conformation was reestablished upon substrate binding. Although it is not easily explicable how peptide binding changes the conformation so dramatically, this result appeared to be in agreement with the crystal structure of Akt2, which was activated by Thr309 phosphorylation and S474D mutation and complexed with GSK3β peptide and AMP-PNP, where the distance between Lys285 and Lys427 was indicated to be 7.0 Å (Protein Data Bank entry 1o6k) (
      • Yang J.
      • Cron P.
      • Thompson V.
      • Good V.M.
      • Hess D.
      • Hemmings B.A.
      • Barford D.
      Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation.
      ).
      In summary, our results strongly suggest that Akt undergoes distinctive conformational changes at each step of activation sequence. Based on our data, the following activation scheme is proposed (Fig. 7). Inactive Akt exists with a folded structure in cytoplasm with the PH and regulatory domains covering the kinase domain at least in part. Membrane translocation causes conformational changes of Akt so that both PH and regulatory domains become separated from the kinase domain to expose Thr308 and Ser473 for phosphorylation. Phosphorylation of Akt presumably triggers the separation of the PH domain from membrane, releasing Akt to the cytoplasm. The open conformation of the regulatory domain in the activated form of cytosolic Akt allows substrate entry, which is an important determinant for the activated state of Akt. This model is consistent with the well accepted notion that Akt activity correlates with conformational changes allowing phosphorylation at Thr308 and Ser473 (
      • Brazil D.P.
      • Hemmings B.A.
      Ten years of protein kinase B signalling: a hard Akt to follow.
      ,
      • Alessi D.R.
      • Cohen P.
      Mechanism of activation and function of protein kinase B.
      ). Furthermore our model may offer an explanation for the previous findings that Ser474 phosphorylation is required for full activation of Akt2 (
      • Yang J.
      • Cron P.
      • Thompson V.
      • Good V.M.
      • Hess D.
      • Hemmings B.A.
      • Barford D.
      Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation.
      ) by depicting the regulatory domain moved away from the kinase domain after phosphorylation, allowing facilitated substrate entry and enhancing overall activity.
      Figure thumbnail gr7
      Fig. 7Schematic model for interdomain conformational changes accompanying Akt activation and substrate binding. In cytosol, inactive Akt exists as a folded molecule with the PH and regulatory domains covering parts of the kinase domain. Akt is recruited to the plasma membrane through the interaction of its PH domain with PIP3. The membrane interaction results in a conformational change, moving the PH and regulatory domains away from the kinase domain and exposing Thr308 and Ser473 for phosphorylation by upstream kinases. When Akt is activated by phosphorylation at both sites, the PH domain closes, possibly enabling the separation of Akt from the membrane. The regulatory domain, which remained open, allows the substrate entry, and upon substrate binding the regulatory domain returns to a closed conformation as in the inactive form. PIP2, phosphatidylinositol 4,5-bisphosphate; PI3K, phosphatidylinositol 3-kinase; pS, phosphoserine; pT, phosphothreonine.
      In conclusion, our results provide the first demonstration for the dramatic interdomain conformational changes in the full-length Akt structure in solution during activation and substrate binding. Our results also provide valuable complementary information to the crystal structures of this molecule. More importantly, our results set up a stage for the further investigation on Akt-membrane, Akt-protein, and/or Akt-drug interactions that is the key to understand the molecular mechanisms involved in physiological and pathophysiological processes of cell survival.

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