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Originally published In Press as doi:10.1074/jbc.M601384200 on March 15, 2006

J. Biol. Chem., Vol. 281, Issue 20, 13949-13956, May 19, 2006
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Kinetic Mechanism of AKT/PKB Enzyme Family*

Xiaoling Zhang{ddagger}1, Shiwen Zhang{ddagger}, Harvey Yamane§, Robert Wahl, Arisha Ali§, Julie A. Lofgren{ddagger}, and Richard L. Kendall{ddagger}

From the Departments of {ddagger}Oncology Research, §Protein Science, and Molecular Pharmacology, Amgen Inc., Thousand Oaks, California 91320

Received for publication, February 13, 2006 , and in revised form, March 15, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
AKT/PKB, the cellular homologue of the murine transforming retrovirus oncogene v-akt (1–5), is a 60-kDa serine/threonine protein kinase composed of three functional domains as follows: a catalytic kinase domain flanked by an N-terminal pleckstrin homology (PH)2 domain and a short hydrophobic C-terminal tail. Three AKT/PKB isoforms, AKT1 (PKB{alpha}), AKT2 (PKBbeta), and AKT3 (PKB{gamma}), (GenBankTM accession numbers NM_005163 [GenBank] , NM_001626 [GenBank] , and NM_005465 [GenBank] ) have been identified with 73% identity (96% similarity) overall and 84% identity (99% similarity) in the kinase domain (6–11). The kinase domain of AKT shares 45% identity (about 60% similarity) with that of protein kinase A and protein kinase C and thus was classified as a member of AGC protein kinase family. The importance of AKT in growth factor signaling was first demonstrated by showing that it is activated by PI 3-kinase in a PH domain-dependent fashion (12–13). Subsequent studies revealed that AKT is activated by the phosphorylation of two critical residues as follows: Thr-308 (AKT1) in the kinase domain and Ser-473 (AKT1) in the C-terminal hydrophobic motif (14). Activation of Akt is triggered by the interaction between the lipid products of PI 3-kinase and the PH domain of AKT, which induces a conformational change in AKT exposing the activation segment of the kinase domain. This serves to recruit the 3-phosphoinositide-dependent protein kinase PDK1 to the proximity of the activation segment allowing the phosphorylation of Thr-308 (Thr-309 for AKT2 and Thr-305 for AKT3) (15–18, 20). The phosphorylation of Thr-308 only partially activates AKT; the phosphorylation of Ser-473 (Ser-474 for AKT2 and Ser-472 for AKT3) of the C-terminal hydrophobic motif completes the transition of AKT from inactive to a fully active conformation and stabilizes the protein in the active state (16–21). Although the Thr-308 kinase has been clearly shown to be PDK1, the identity of the kinase that phosphorylates Ser-473 has been in dispute. Recent studies suggested several candidate enzymes, including DNA-PKcs, ATM, and the mTOR-Rictor complex (22–25). Interestingly, all three enzymes belong to the PI 3-kinase superfamily, which is consistent with the fact that the phosphorylation of Ser-473 is inhibited by the PI 3-kinase inhibitor wortmannin and LY294002. It is possible that the regulation of Ser-473 involves multiple activating complexes under different conditions and therefore offers another level of specificity in signaling.

The physiological functions of AKT are executed through a wide range of downstream effectors involved in cell survival, cell proliferation, and cell growth (for reviews see Refs. 26–29). These substrates all share a common signature motif RXRXX(S/T), where the S/T is the phosphorylation site (27, 30). A peptide library screening approach has further defined the preferred residues flanking the phosphorylation site (31). The studies of individual isoforms of AKT have not shown obvious substrate specificity among the isoforms (19, 32). However, genetic deletion experiments in mice have demonstrated distinct roles of AKT1, AKT2, and AKT3 (33–36). One possible explanation for the isoform-specific functions could simply be the differential tissue expression pattern of the AKT isoforms because tissues types where the major defect resides in the AKT1, AKT2, and AKT3 knock-out mice somewhat correlate with the expression of a particular AKT isoform. However, isoform-specific phenotypes could not be completely explained by the tissue-specific expression pattern. For example, overexpression of AKT1 in adipocytes derived from AKT2–/– mouse embryo fibroblast cells was not able to rescue insulin response (37). In addition, although both AKT1 and AKT3 gene deletion in mice led to a brain size reduction, AKT1 deleted mice only exhibited decreased cell number, whereas AKT3 deleted mice exhibited both decreased cell number and volume (36).

Although AKT has been extensively studied in the last decade, the kinetic mechanism of its intrinsic kinase activity has not been described in detail. Thus there is little understanding of whether the enzyme catalytic properties of AKT contribute to the isoform-specific functions. One of the first detailed kinetic mechanistic studies on a protein kinase was described by Cook et al. (38) and Whitehouse et al. (39). The prototype kinase in these studies was cAMP-dependent protein kinase (PKA), an AGC family kinase with 50% homology in the kinase domain and structural similarity to AKT (20–21). Both studies showed that the PKA kinase reaction follows a sequential mechanism by which a ternary complex is formed prior to the product release. However, the order of substrate addition has been in dispute. The study by Whitehouse et al. (39) concluded that the substrate addition follows an ordered kinetic mechanism with ATP binding first to the enzyme, although Cook and co-workers (38, 40, 41) suggested that the substrate addition is random with ordered release of products. In this study we have generated activated AKT1, AKT2, and AKT3 and investigated their kinetic mechanism against an optimal synthetic peptide substrate. We have also obtained kinetic parameters of AKT1, AKT2, and AKT3 in their cellular complex to verify the results from in vitro purified enzymes. The implications of our results are further discussed.


Figure 1
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  		FIGURE 1.
Characterization of AKT enzymes used in the study. A, AKT1, AKT2, AKT3 were activated and purified in vitro as described under "Experimental Procedures." Aliquots of 0.5 and 0.1 µg of proteins were analyzed by SDS-PAGE and stained by SimplyBlueTM SafeStain (catalogue number LC6060; Invitrogen). The positions of molecular weight markers were labeled to the left. B, Western analysis of the level of phospho-Ser-473 and phospho-Thr-308 of the same set of AKT isoforms as in A. Aliquots of 1, 2, and 5 ng from each isoform were loaded to SDS-PAGE, transferred to nitrocellulose, and blotted by phospho-Ser-473, phospho-Thr-308, and pan-AKT antibodies. C, Western blots of immunoprecipitates from Rat-1 cell lines expressing myr-HA-AKT1, myr-HA-AKT2, myr-HA-AKT3, and vector. Aliquots of 150 µg of total protein lysates from each cell line were immunoprecipitated by HA-monoclonal antibody. Each immunoprecipitate was split into 2 aliquots in a ratio of 1:3 and run by SDS-PAGE. The blots were detected by phospho-Ser-473, phospho-Thr-308, and pan-AKT antibodies.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—ATP, ADP, and AMP-PCP were purchased from Sigma. [{gamma}-33P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. All peptides were synthesized by the Amgen Peptide Technology Group. PIP3 was purchased from AG Scientific. pbabePuroL plasmids containing myr-HA-AKT1, myr-HA-AKT2, and myr-AKT3 cDNAs were gifts from Dr. William R. Sellers (Dana-Farber Cancer Institute, Harvard Medical School) (42, 43). PDK1 and MAPKAPK-2 were generated by Amgen. The 96-well MultiScreen® phosphocellulose filter plates (catalogue number MAPH N0B) were from Millipore. The 96-well Reacti-BindTM protein G plates (catalogue number 15131) were from Pierce. Anti-AKT (catalogue number 9272) and antiphospho-AKT (catalogue numbers 9271 and 9275) antibodies were from Cell Signaling Technology. Anti-HA antibody (12CA5) was from Roche Applied Science (catalogue number 11583816). Cell culture reagents were from Invitrogen. All other general reagents were from Sigma.

Enzyme Preparation—Full-length AKT1, AKT2, and AKT3 cDNA were cloned into pFastBacHT baculovirus expression vector (catalogue number 10584-027; Invitrogen), which added a His6 tag to the N terminus of the AKT sequence. Recombinant baculoviruses expressing the AKT cDNAs were generated using the Bac-to-Bac baculovirus expression system (Invitrogen) and used to infect Sf9 cells to produce cell pastes with recombinant AKT proteins. For all three isoforms, insect cell paste (60–100 g) was resuspended in 5 ml of lysis buffer per g (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM imidazole, 10% glycerol, 5 mM DTT, 1x Complete (Roche Applied Science)) and lysed by nitrogen cavitation in a Parr bomb. Lysates were clarified by centrifugation at 20,000 x g followed by 0.45-µm filtration. The filtered lysates were batch bound to Talon (Clontech), washed, and eluted with lysis buffer containing 100 mM imidazole. For Akt3 and Akt1, Talon elution fractions were applied to a HiTrap Q HP column (Amersham Biosciences) and eluted with a linear gradient of 0–0.5 M NaCl. For Akt1, peak fractions were concentrated and further purified on a Superdex 200 XK 26/60 column (Amersham Biosciences). For Akt2, Talon elution fractions were applied to a HiTrap Blue column (Amersham Biosciences) and eluted with a linear gradient of 0–2 M NaCl. Fractions containing Akt2 were pooled, diluted, and applied to a HiTrap Q HP column and eluted with a linear gradient of 0–0.5 M NaCl. Peak fractions were pooled, concentrated, and further purified on a Superdex 200 XK 26/60 column.

Each isoform of Akt was activated by PDK1 and MAPKAPK-2 as follows. Lipid vesicles were prepared by drying 200 µg (0.17 µmol) of PIP3 (AG Scientific), 1.68 µmol of PC, and 1.68 µmol of PS (Avanti%20Polar%20Lipids">Avanti Polar Lipids) in a siliconized glass tube in a SpeedVac. Lipids were resuspended in 1.68 ml of activation buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT) and freeze-thawed three times. The resulting suspension was extruded 15 times through a 0.1-µm membrane in a mini-extruder (Avanti). Akt (4 mg), PDK1 (0.4 mg), and p38-activated MAPKAPK-2 (0.4 mg) were added to 1.6 ml of PIP3/PC/PS vesicles. ATP and MgCl2 were added to 1 and 10 mM, respectively, and the final reaction volume was adjusted to 8 ml with activation buffer. The activation reaction was allowed to proceed for 1 h at room temperature. Reactions were quenched by the addition of 1/19 volume of 20% Triton X-100 (1% final) and then centrifuged at 100,000 x g for 1 h. The supernatants were diluted and applied to a HiTrap Q HP column and eluted with a linear gradient of 0–0.5 M NaCl. Peak fractions were pooled, and protein concentration was determined by the method of Bradford using bovine serum albumin as a standard.

AKT Kinase Reaction—The kinase activity of AKT was measured by phosphorylating an optimal AKT peptide substrate identified from peptide library screening by Obata et al. (31) as follows: ARKRERTYS*FGHHA, where S* is the phosphorylation site. The reaction mixture contained 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM DTT, 1 mM EGTA, 1 mM Na3VO4, 0.1% bovine serum albumin (kinase reaction buffer), with the addition of activated AKT. The kinase reactions were performed ranging from 5 to 20 min and enzyme concentrations from 0.4 to 3.3 nM to determine linearity. The optimal concentration of AKT1, AKT2, and AKT3 in the reactions was determined to be 1.7, 3.3, and 1.7 nM, respectively. The reactions were initiated by adding the peptide substrate and [{gamma}-33P]ATP/ATP in a final reaction volume of 100 µl. The reaction mixtures were incubated at room temperature for 10 min and stopped by adding 100 µl of 5% phosphoric acid. Under these conditions, all initial velocity reactions utilized less than 10% of the substrates. For the measurement of the products, the total mixtures were transferred to phosphocellulose filter plates. The peptide substrates were captured by the phosphocellulose filter paper after vacuum application and washed three times by 1% phosphoric acid. The filter plates were counted by Wallac MicroBeta® liquid scintillation counter (PerkinElmer Life Sciences).


Figure 2
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  		FIGURE 2.
Two-substrate kinetic analysis of AKT1, AKT2, and AKT3 enzymes. Double-reciprocal plots of 1/v versus 1/ATP (left) and 1/v versus 1/peptide (right) from initial velocity studies. Conditions were as described under "Experimental Procedures." A, AKT1 velocities were measured in the presence of 7.8–250 µM ATP and 0.23–7.5 µM of peptide substrate; B, AKT2 velocities were measured in the presence of 15.6–500 µM ATP and 0.47–30 µM of peptide substrate; C, AKT3 velocities were measured in the presence of 15.6–500 µM ATP and 3.125–100 µM of peptide substrate.

 
Kinase Reaction of Immunoprecipitated AKT—Rat-1 fibroblast cells stably expressing myr-HA-AKT1, myr-HA-AKT2, and myr-HA-AKT3 were generated using pbabePuroL-AKT expression plasmids and were expanded in Dulbecco's modified Eagle's media containing 10% fetal bovine serum, 2 mM L-glutamine, and 3 µg/ml puromycin (selective agent). To prepare for the cell lysate, cells were washed by PBS and lysed on ice by the addition of a lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerol phosphate, 1 mM Na3VO4, 1 µM microcystin-LR, and 1x EDTA-free protease inhibitor mixture (catalogue number 1873580; Roche Applied Science). Clear lysate was isolated from cell debris by a refrigerated bench-top centrifuge at 14,000 x g for 15 min. To specifically pull down the myr-HA-AKT complex, lysates were diluted to a buffer containing 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl (TBS) at a 1:1 ratio, applied to a Reacti-BindTM protein G plate coated with anti-HA antibody, allowed to bind for 1.5 h at room temperature, and washed three times in TBS with 0.2% Triton X-100 followed by a final wash with kinase reaction buffer. The kinase reactions were initiated by adding 50 µl of kinase reaction buffer containing peptide and [{gamma}-33P]ATP/ATP in the indicated amount. The reaction was incubated at room temperature for 10 min and stopped and counted as described above.

Data Analysis—Kinetic data were fitted to equations using the iterative minimum {chi}2 nonlinear regression method of Leatherbarrow (44) and converted into double-reciprocal Lineweaver-Burk plots. All kinetic studies were performed in duplicate when using purified enzymes and in quadruplicate when using the immunoprecipitated enzymes. The equations for kinetics analysis were as described by Cleland (45–47) and Segel (48).

For single substrate kinetics measurements (Equation 1),

Formula 1(Eq. 1)

For the discrimination between sequential and ping-pong mechanism of two-substrate kinetics, data were fitted into Equations 2 and 3,

Formula 2(Eq. 2)
and

Formula 3(Eq. 3)
For the determination of enzyme inhibition mechanism, data were fitted into Equations 4, 5, 6,

Formula 4(Eq. 4)

Formula 5(Eq. 5)
and

Formula 6(Eq. 6)
In these equations, v represents the measured velocity; Vmax is the maximum velocity; A, B, and S are substrates; Ka, Kb, and Km are the Michaelis constants for A, B, and S; Kia is the dissociation constant for A; Kib is the dissociation constant for B; and Ki is the inhibitor constant.

For the determination of interaction between the two substrate sites, data were fitted into Equation 7,

Formula 7(Eq. 7)
where the {alpha} factor indicates whether or not the two sites are independent (48). When {alpha} = 1, there is no interaction, and the two substrates are independent. When {alpha} < 1, the two substrate sites interact with each other with positive cooperativity; the binding of one substrate increases the affinity of the other substrate. When {alpha} > 1, the two substrate sites interact with each other with negative cooperativity; the binding of one substrate decreases the affinity of the other substrate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Initial Velocity Studies—The three AKT isoforms AKT1, AKT2, and AKT3 were His-tagged and expressed in an Sf9 cell baculovirus system and purified to homogeneity. The AKT proteins were activated by PDK1 in the presence of PIP3 and subsequently by MAPKAPK-2 as described under "Experimental Procedures." The reactions were driven to saturation by excess ATP to allow maximum activation of AKT. The activated AKT proteins were re-purified, quantified, and examined by Western analysis as shown in Fig. 1. All three isoforms were purified to greater than 90% homogeneity (Fig. 1A). The Thr-308 and Ser-473 sites were phosphorylated to similar levels across the isoforms with a subtle difference as follows: the highest percentage being in AKT2 followed by AKT1 and AKT3 (Fig. 1B).


Figure 3
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  		FIGURE 3.
Product inhibition of AKT1, AKT2, and AKT3 enzymes by ADP. A, AKT1, double-reciprocal plots of 1/v versus 1/ATP with 7.8–250 µM ATP and 20 µM peptide (left); and 1/v versus 1/peptide with 0.47–7.5 µM peptide and 20 µM ATP (right). B, AKT2, double-reciprocal plots of 1/v versus 1/ATP with 15.6–500 µM ATP and 20 µM peptide (left); and 1/v versus 1/peptide with 0.94–15 µM peptide and 200 µM ATP (right). C, AKT3, double-reciprocal plots of 1/v versus 1/ATP with 15.6–500 µM ATP and 20 µM peptide (left); and 1/v versus 1/peptide with 0.94–15 µM peptide and 100 µM ATP (right). ADP concentrations were as indicated. Conditions were as described under "Experimental Procedures."

 
Two-substrate site kinetics of the forward reactions was performed using these enzymes in the absence of added products. Initial velocities were measured in a matrix of varied ATP concentration and peptide substrate concentrations, and the data were fitted to the equations for sequential and ping-pong mechanism (see Equations 2 and 3 under "Experimental Procedures") to distinguish between the two mechanisms. The double-reciprocal plots of AKT1, AKT2, and AKT3 two-substrate kinetics all best fitted to the pattern of a typical sequential mechanism where the plots of one substrate at varied concentrations of the other one converged to the left of the y axis (Fig. 2), whereas if the reactions followed the ping-pong mechanism, the plots should have been parallel lines with the same slope factors. These results suggest that the catalysis of AKT enzymes requires the formation of a ternary complex of the enzyme with the two substrates before product release. The kinetic constants obtained from the two-substrate kinetic studies were summarized in Table I, part A.


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  		TABLE 1
Comparison of the Kinetic constants of AKT1, AKT2 and AKT3

AKT represents purified and in vitro activated AKT isoforms, and myr-AKT-IP represents recombinant myristoylated AKT isoforms expressed and immunoprecipitated from Rat-1 cells. Peptide = ARKRERTYSFGHHA. The experimental details and data analysis are as described under "Experimental Procedures."

 


Figure 4
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  		FIGURE 4.
Dead-end inhibition of AKT1 by Ala peptide. Double-reciprocal plots of 1/v versus 1/ATP with 15.6–250 µM ATP and 2 µM peptide (top); and 1/v versus 1/peptide with 0.94–15 µM peptide and 50 µM ATP (bottom). Ala peptide concentrations were as indicated. Conditions were as described under "Experimental Procedures."

 
The double-reciprocal lines did not converge exactly on the x axis, suggesting that binding of one substrate affects the binding of the other substrate, i.e. the two substrate sites are interactive. This finding is similar to what has been observed for PKA in its kinetic study (39) and the crystal structure study with protein kinase inhibitor (PKI) and ATP (49). The two-site interaction factor {alpha} was determined using the rapid equilibrium sequential model (see "Experimental Procedures," Equation 7; Table 1, part A). In the case of AKT1 and AKT3, binding of one substrate increased the binding of the other substrate. However, for AKT2, the binding of one substrate decreased the binding of the other substrate, although the degree of interaction was within 2-fold. It is important to note that the interaction factor was determined from purified recombinant proteins tested in vitro; it is possible that the direction and the degree of the interaction can be modulated by other AKT-interacting proteins, possibly providing a mechanism for the feedback regulation of AKT activity.

Unexpected from the high degree of homology in the kinase domains, different AKT isoforms showed different kinetic parameters. AKT2 exhibited the greatest dissociation constant and apparent Km value for ATP compared with AKT1 and AKT3. However, AKT3 showed the highest catalytic efficiency with a kcat of 119, whereas the kcat values of AKT1 and AKT2 were similar (22 for AKT1 and 25 for AKT2). To further confirm that the differences of the AKT isoforms in their kinetic constants are relevant to their activated state in situ, immunoprecipitates of constitutively active AKT1, AKT2, and AKT3 expressed in Rat-1 fibroblast cells (myr-HA-AKTs) were used to obtain the Km values for ATP and peptide substrate. The three myr-HA-AKT isoforms were all dually phosphorylated at Thr-308 and Ser-473 sites as shown in Fig. 1C. Consistent with the results from purified enzymes, AKT2 also showed the highest Km value for ATP (Table 1, part B), suggesting that the three AKT isoforms are different in their intrinsic kinetic properties.


Figure 5
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  		FIGURE 5.
Dead-end inhibition of AKT1 by AMP-PCP. Double-reciprocal plots of 1/v versus 1/ATP with 31.25–500 µM ATP and 10 µM peptide (top) and 1/v versus 1/peptide with 0.94–15 µM peptide and 50 µM ATP (bottom). Ala peptide concentrations were as indicated. Conditions were as described under "Experimental Procedures."

 
Product Inhibition—To distinguish between a random or ordered substrate addition mechanism for the AKT two-substrate kinetics, initial velocities of AKT isoforms were measured in the presence of the reaction product ADP (Fig. 3). ADP inhibited the reactions of all three AKT isoforms with a similar pattern in which ADP was a competitive inhibitor versus ATP and a noncompetitive inhibitor versus peptide. This pattern is the same as in PKA reactions as shown by Whitehouse et al. (39) and is consistent with a steady state Ordered Bi Bi mechanism in which the ATP is the first substrate to bind to the enzyme, and ADP is the last product released. However, if the two substrates bind to the enzyme randomly, the inhibition would have been noncompetitive versus both ATP and peptide (39, 48). In addition, these data suggest that under the conditions tested the three AKT isoforms share the same kinetic mechanism, and thus AKT1 was used in the subsequent studies as a model to further elucidate the steady state kinetics of the AKT enzymes.

Dead-end Inhibition—Dead-end inhibitors often provide definitive answers in discriminating among the different kinetic mechanisms. Two dead-end inhibitors were used in this study to confirm the results from product inhibition as follows: AMP-PCP, a nonhydrolyzable ATP analogue, and Ala peptide, the substrate peptide with the phosphate acceptor residue serine altered to alanine. When the peptide was the varied substrate, the pattern of the double-reciprocal lines at different concentrations of Ala peptide converged to the y axis (Fig. 4), suggesting that Ala peptide is a competitive inhibitor of the peptide substrate and the Ala peptide shares the binding site with the peptide substrate exclusively. When ATP was the varied substrate, the Ala peptide behaves as an uncompetitive inhibitor where the double-reciprocal lines at different concentrations of Ala peptide were parallel to each other (Fig. 4). These data are consistent with a mechanism where the peptide substrate site is only available when ATP is complexed with the enzyme, i.e. the binding of peptide substrate to the enzyme requires the presence of ATP. If the binding of the two substrates were random, the inhibition pattern would have been noncompetitive versus ATP. These results provided strong support for an Ordered Bi Bi kinetic mechanism where ATP binding to the enzyme is a prerequisite for peptide substrate binding. The second dead-end inhibitor ATP analogue AMP-PCP inhibited the reaction as a competitive inhibitor when ATP was the varied substrate and a noncompetitive inhibitor when peptide was the varied substrate (Fig. 5). These data further confirmed that ATP is the first substrate to bind to the AKT enzymes and thus the Ordered Bi Bi kinetic mechanism for AKT. The patterns of inhibition and kinetic constants of the product and dead-end inhibition study of AKT1 are summarized in Table 2. Therefore, the data taken together were consistent with the kinetic scheme as illustrated in Scheme 1.


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  		TABLE 2
Kinetic constants of AKT1 from product and dead-end inhibition

Purified and in vitro activated AKT1 was used for this study. The abbreviations used are as follows: C, competitive; NC, noncompetitive; UC, uncompetitive. The experimental details and data analysis are as described under "Experimental Procedures."

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our initial velocity studies, product inhibition, and dead-end inhibition have consistently shown that AKT enzymes follow an ordered sequential kinetic mechanism by which ATP binds to the enzyme first and protein substrate binding is dependent on the ATP binding, which has also been shown for PKA, another member of AGC family (39). This kinetic mechanism suggests that the ATP, rather than protein substrate of AKTs, initiates the AKT reaction and perhaps provides a rate-limiting step in the regulation of its activity. One argument against this hypothesis can be that the cellular ATP concentration is too high (considered in the low millimolar range) and that the magnitude of ATP concentration change in cells can hardly impact the enzyme activity of a kinase. Therefore studies investigating the cellular concentrations of ATP and the product inhibitor ADP under the physiological conditions will allow further testing of this hypothesis. It is possible that cells in tissues under low oxygen tension have a much lower ATP level than what is measured in cell cultures where excess oxygen and glucose are supplemented. Therefore, the ATP level in tissues under hypoxic conditions may set the threshold for AKT activity and thus the survival signal. In fact, there is increasing evidence that AKT activity is regulated by ATP levels in cells. A study by Hresko et al. (50) showed that reduced intracellular ATP by glucosamine treatment impaired insulin response. Although the study did not directly measure AKT kinase activity in response to ATP reduction, it is likely that the effect of ATP depletion on insulin response was through its major downstream target AKT. Recent studies have also shown that the activity of AKT and another kinase in the same pathway mTOR is dependent on the glucose level and the mitochondrial hexokinase activity, and both play key roles in the regulation of cellular ATP levels (51–53). Perhaps more direct evidence is available from a study by Nagata et al. (54) examining the role of AMP-activated protein kinase (AMPK) in the angiogenic activity of endothelial cells under hypoxic conditions. AMPK is an ATP sensor kinase that increases the cellular ATP level in responses to hypoxia and other cellular energy stress. The study showed that AMPK was required for vascular endothelial growth factor-stimulated AKT kinase activity only under hypoxia conditions but not under normoxia conditions, suggesting that AKT activity is dependent on the cellular ATP level, which is maintained by AMPK under hypoxia. Recent studies also showed that AKT can in turn increase cellular ATP levels (53, 55), suggesting that the cellular energy state and ATP level can provide a positive feedback loop to drive the AKT activity.


Figure 6
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  		SCHEME 1
 
Although the three AKT isoforms seem to share the same kinetic mechanism, the kinetic constants of these isoforms were different thus suggesting different catalytic properties among the isoforms. The difference was mainly associated with the affinity of the isoforms for ATP and the catalytic efficiency. AKT2 showed the lowest affinity for ATP and thus about a 5-fold higher Km value than that of AKT1 and AKT3. Interestingly, the physiological role of AKT2 is also distinct from that of AKT1 and AKT3 in that AKT2 has been shown to be the major effector in insulin signaling and glucose metabolism, whereas AKT1 and AKT3 are mainly involved in growth and survival with AKT1 functioning in all organs and AKT3 specifically in the brain (33–36). The high expression of AKT2 in insulin-responsive tissues has not been able to completely explain this functional difference (37). It is possible that AKT2 is adapted to these specific tissues in such way that through a dependence on its catalytic properties, only this isoform is able to sense a change in ATP concentration and thus glucose metabolism. AKT3, the lowest abundant isoform of the three, showed the highest catalytic activity with a 5-fold higher kcat value than AKT1 and AKT2. Genetic deletion of AKT3 in mice led to a specific brain size reduction (36). Protein expression analysis in mouse brain showed that AKT3 represents 50% of total AKT activity, whereas AKT1 and AKT2 make up the other 50% of the activity (36). It will be interesting to test whether simply elevated expression of AKT1 or AKT2 can compensate for the loss of AKT3 activity in brain cells or if there is a specific requirement for AKT3.

AKT resides in the central node of receptor signaling and has been demonstrated to be a critical factor in both normal and tumor cell function. The fact that AKT enzyme activity is constitutively activated in a wide range of cancers has positioned AKT as an attractive drug target for cancer therapy. Distinct functions of AKT isoforms shown by mouse genetic studies have suggested that an isoform-selective AKT inhibitor can be efficacious on selected tumor types. The different intrinsic catalytic properties of these isoforms may provide an opportunity for the development of isoform-selective kinase inhibitors of AKT.


   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. Back

1 To whom correspondence should be addressed: Dept. of Oncology Research, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320. Tel.: 805-447-1362; Fax: 805-499-2751; E-mail: xzhang{at}amgen.com.

2 The abbreviations used are: PH, pleckstrin homology; AMP-PCP, 5'-phosphoadenosine {alpha},beta-methylene diphosphonate; DTT, dithiothreitol; PIP3, inositol 1,4,5-trisphosphate; myr, myristoylated; HA, hemagglutinin; PKA, cAMP-dependent protein kinase; AMPK, AMP-activated protein kinase; PI, phosphatidylinositol. Back


   ACKNOWLEDGMENTS
 
We thank the peptide technology group at Amgen for synthesizing the peptides used in this study, Dr. William R. Sellers for providing us the Rat-1-AKT cell lines, and Dr. Murray Robinson for helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Staal, S. P., Hartley, J. W., and Rowe, W. P. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3065–3067[Abstract/Free Full Text]
  2. Staal, S. P. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5034–5037[Abstract/Free Full Text]
  3. Jones, P. F., Jakubowicz, T., Fernando, J. P., Maurer, F., and Hemmings, B. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4171–4175[Abstract/Free Full Text]
  4. Bellacosa, A., Testa, J. R., Staal, S. P., and Tsichlis, P. N. (1991) Science 254, 274–277[Abstract/Free Full Text]
  5. Coffer, P. J., and Woodgett, J. R. (1991) Eur. J. Biochem. 201, 475–481[Medline] [Order article via Infotrieve]
  6. Jones, P. F., Jakubowicz, T., and Hemmings, B. A. (1991) Cell Regul. 2, 1001–1019[Medline] [Order article via Infotrieve]
  7. Cheng, J. Q., Godwin, A. K., Bellacosa, A., Taguchi, T., Franke, T. F., Hamilton, T. C., Tsichlis, P. N., and Testa, J. R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9267–9271[Abstract/Free Full Text]
  8. Konishi, H., Kuroda, S., Tanaka, M., Matsuzaki, H., Ono, Y., Kameyama, K., Haga, T., and Kikkawa, U. (1995) Biochem. Biophys. Res. Commun. 216, 526–534[CrossRef][Medline] [Order article via Infotrieve]
  9. Nakatani, K., Sakaue, H., Thompson, D. A., Weigel, R. J., and Roth, R. A. (1999) Biochem. Biophys. Res. Commun. 257, 906–910[CrossRef][Medline] [Order article via Infotrieve]
  10. Brodbeck, D., Cron, P., and Hemmings, B. A. (1999) J. Biol. Chem. 274, 9133–9136[Abstract/Free Full Text]
  11. Masure, S., Haefner, B., Wesselink, J. J., Hoefnagel, E., Mortier, E., Verhasselt, P., Tuytelaars, A., Gordon, R., and Richardson, A. (1999) Eur. J. Biochem. 265, 353–360[Medline] [Order article via Infotrieve]
  12. Franke, T. F., Yang, S. I., Chan, T. O., Datta, K., Kazlauskas, A., Morrison, D. K., Kaplan, D. R., and Tsichlis, P. N. (1995) Cell 81, 727–736[CrossRef][Medline] [Order article via Infotrieve]
  13. Burgering, B. M., and Coffer, P. J. (1995) Nature 376, 599–602[CrossRef][Medline] [Order article via Infotrieve]
  14. Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., and Hemmings, B. A. (1996) EMBO J. 15, 6541–6551[Medline] [Order article via Infotrieve]
  15. Franke, T. F., Kaplan, D. R., Cantley, L. C., and Toker, A. (1997) Science 275, 665–668[Abstract/Free Full Text]
  16. Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., and Cohen, P. (1997) Curr. Biol. 7, 261–269[CrossRef][Medline] [Order article via Infotrieve]
  17. Stokoe, D., Stephens, L. R., Copeland, T., Gaffney, P. R., Reese, C. B., Painter, G. F., Holmes, A. B., McCormick, F., and Hawkins, P. T. (1997) Science 277, 567–570[Abstract/Free Full Text]
  18. Stephens, L., Anderson, K., Stokoe, D., Erdjument-Bromage, H., Painter, G. F., Holmes, A. B., Gaffney, P. R., Reese, C. B., McCormick, F., Tempst, P., Coadwell, J., and Hawkins, P. T. (1998) Science 279, 710–714[Abstract/Free Full Text]
  19. Walker, K. S., Deak, M., Paterson, A., Hudson, K., Cohen, P., and Alessi, D. R. (1998) Biochem. J. 331, 299–308[Medline] [Order article via Infotrieve]
  20. Yang, J., Cron, P., Thompson, V., Good, V. M., Hess, D., Hemmings, B. A., and Barford, D. (2002) Mol. Cell 9, 1227–1240[CrossRef][Medline] [Order article via Infotrieve]
  21. Yang, J., Cron, P., Good, V. M., Thompson, V., Hemmings, B. A., and Barford, D. (2002) Nat. Struct. Biol. 9, 940–944[CrossRef][Medline] [Order article via Infotrieve]
  22. Feng, J. H., Park, J., Cron, P., Hess, D., and Hemmings, B. A. (2004) J. Biol. Chem. 279, 41189–41196[Abstract/Free Full Text]
  23. Dragoi, A. M., Fu, X. Y., Ivanov, S., Zhang, P., Sheng, L. B., Wu, D. Q., Li, G. C., and Chu, W. M. (2005) EMBO J. 24, 779–789[CrossRef][Medline] [Order article via Infotrieve]
  24. Viniegra, J. G., Martinez, N., Modirassari, P., Losa, J. H., Cobo, C. P., Lobo, V. J. S. A., Luquero, C. I. A., Alvarez-Vallina, L., Cajal, S. R. Y., Rojas, J. M., and Sanchez-Prieto, R. (2005) J. Biol. Chem. 280, 4029–4036[Abstract/Free Full Text]
  25. Sarbassov, D. D, Guertin, D. A., Ali, S. M., and Sabatini, D. M. (2005) Science 307, 1098–1101[Abstract/Free Full Text]
  26. Datta, S. R., Brunet, A., and Greenberg, M. E. (1999) Genes Dev. 13, 2905–2927[Free Full Text]
  27. Lawlor, M. A., and Alessi, D. R. (2001) J. Cell Sci. 114, 2903–2910[Medline] [Order article via Infotrieve]
  28. Manning, B. D., and Cantley, L. C. (2003) Biochem. Soc. Trans. 31, 573–578[CrossRef][Medline] [Order article via Infotrieve]
  29. Brazil, D. P., Yang, Z. Z., and Hemmings, B. A. (2004) Trends Biochem. Sci. 29, 233–242[CrossRef][Medline] [Order article via Infotrieve]
  30. Alessi, D. R., and Cohen, P. (1998) Curr. Opin. Genet. Dev. 8, 55–62[CrossRef][Medline] [Order article via Infotrieve]
  31. Obata, T., Yaffe, M. B., Leparc, G. G., Piro, E. T., Maegawa, H., Kashiwagi, A., Kikkawa, R., and Cantley, L. C. (2000) J. Biol. Chem. 275, 36108–36115[Abstract/Free Full Text]
  32. Meier, R., Alessi, D. R., Cron, P., Andjelkovic, M., and Hemmings, B. A. (1997) J. Biol. Chem. 272, 30491–30497[Abstract/Free Full Text]
  33. Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., III, Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001) Science 292, 1728–1731[Abstract/Free Full Text]
  34. Chen, W. S., Xu, P. Z., Gottlob, K., Chen, M. L., Sokol, K., Shiyanova, T., Roninson, I., Weng, W., Suzuki, R., Tobe, K., Kadowaki, T., and Hay, N. (2001) Genes Dev. 15, 2203–2208[Abstract/Free Full Text]
  35. Cho, H., Thorvaldsen, J. L., Chu, Q., Feng, F., and Birnbaum, M. J. (2001) J. Biol. Chem. 276, 38349–38352[Abstract/Free Full Text]
  36. Easton, R. M., Cho, H., Roovers, K., Shineman, D. W., Mizrahi, M., Forman, M. S., Lee, V. M. Y., Szabolcs, M., de Jong, R., Oltersdorf, T., Ludwig, T., Efstratiadis, A., and Birnbaum, M. J. (2005) Mol. Cell. Biol. 25, 1869–1878[Abstract/Free Full Text]
  37. Bae, S. S., Cho, H., Mu, J., and Birnbaum, M. J. (2003) J. Biol. Chem. 278, 49530–49536[Abstract/Free Full Text]
  38. Cook, P. F., Neville, M. E., Jr., Vrana, K. E., Hartl, F. T., and Roskoski, R., Jr. (1982) Biochemistry 21, 5794–5799[CrossRef][Medline] [Order article via Infotrieve]
  39. Whitehouse, S., Feramisco, J. R., Casnellie, J. E., Krebs, E. G., and Walsh, D. A. (1983) J. Biol. Chem. 258, 3693–3701[Abstract/Free Full Text]
  40. Qamar, R., Yoon, M. Y., and Cook, P. F. (1992) Biochemistry 31, 9986–9992[CrossRef][Medline] [Order article via Infotrieve]
  41. Qamar, R., and Cook, P. F. (1993) Biochemistry 32, 6802–6806[CrossRef][Medline] [Order article via Infotrieve]
  42. Ramaswamy, S., Nakamura, N., Vazquez, F., Batt, D. B., Perera, S., Roberts, T. M., and Sellers, W. R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2110–2115[Abstract/Free Full Text]
  43. Nakamura, N., Ramaswamy, S., Vazquez, F., Signoretti, S., Loda, M., and Sellers, W. R. (2000) Mol. Cell. Biol. 20, 8969–8982[Abstract/Free Full Text]
  44. Leatherbarrow, R. J. (2001) GraFit, Version 5, Erithacus Software Ltd., Horley, UK
  45. Cleland, W. W. (1963) Biochim. Biophys. Acta 8, 104–137[Medline] [Order article via Infotrieve]
  46. Cleland, W. W. (1963) Biochim. Biophys. Acta 12, 173–187
  47. Cleland, W. W. (1963) Biochim. Biophys. Acta 12, 188–196
  48. Segel, I. H. (1993) Enzyme Kinetics, John Wiley & Sons, Inc., New York
  49. Zheng, J., Knighton, D. R., ten Eyck, L. F., Karlsson, R., Xuong, N., Taylor, S. S., and Sowadski, J. M. (1993) Biochemistry 32, 2154–2161[CrossRef][Medline] [Order article via Infotrieve]
  50. Hresko, R. C., Heimberg, H., Chi, M. M. Y., and Mueckler, M. (1998) J. Biol. Chem. 273, 20658–20668[Abstract/Free Full Text]
  51. Dennis, P. B., Jaeschke, A., Saitoh, M., Fowler, B., Kozma, S. C., and Thomas, G. (2001) Science 294, 1102–1105[Abstract/Free Full Text]
  52. Gottlob, K., Majewski, N., Kennedy, S., Kandel, E., Robey, R. B., and Hay, N. (2001) Genes Dev. 15, 1406–1418[Abstract/Free Full Text]
  53. Majewski, N., Nogueira, V., Bhaskar, P., Coy, P. E., Skeen, J. E., Gottlob, K., Chandel, N. S., Thompson, C. B., Robey, R. B., and Hay, N. (2004) Mol. Cell 16, 819–830[CrossRef][Medline] [Order article via Infotrieve]
  54. Nagata, D., Mogi, M., and Walsh, K. (2003) J. Biol. Chem. 278, 31000–31006[Abstract/Free Full Text]
  55. Hahn-Windgassen, A., Nogueira, V., Chen, C. C., Skeen, J. E., Sonenberg, N., and Hay, N. (2005) J. Biol. Chem. 280, 32081–32089[Abstract/Free Full Text]

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