text:
Acidic Amino Acids Impart Enhanced Ca2+ Permeability and Flux in Two Members of the ATP-gated P2X Receptor Family

P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca2+ permeability of the P2X2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca2+ currents (Pf%) of the rat and human P2X1 and P2X4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X1 receptor but had no effect on the lower Pf% of the P2X2 receptor, suggesting that ionized side chains regulate the Ca2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X1 and P2X4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X2 receptor. Taken together, the data suggest that COO− side chains provide an electrostatic force that interacts with Ca2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X1 and P2X4 receptors is conserved in the P2X3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His45 increased Pf% of the P2X3 channel, suggesting that this positive charge masks the facilitation of Ca2+ flux by the neighboring Glu46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca2+ permeability and flux of some P2X receptors.

P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca 2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca 2+ permeability of the P2X 2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca 2+ currents (Pf%) of the rat and human P2X 1 and P2X 4 subtypes. We used patchclamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X 1 receptor but had no effect on the lower Pf% of the P2X 2 receptor, suggesting that ionized side chains regulate the Ca 2+ fl ux of some family members. Removing the fi xed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X 1 and P2X 4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X 2 receptor. Taken together, the data suggest that COO − side chains provide an electrostatic force that interacts with Ca 2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X 1 and P2X 4 receptors is conserved in the P2X 3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His 45 increased Pf% of the P2X 3 channel, suggesting that this positive charge masks the facilitation of Ca 2+ fl ux by the neighboring Glu 46 . The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca 2+ permeability and fl ux of some P2X receptors.

I N T R O D U C T I O N
A change in calcium concentration is an essential intracellular signal that initiates fundamental physiological processes such as secretion and contraction. To succeed in this role, the intracellular free Ca 2+ concentration ([Ca 2+ ] i ) is tightly controlled, and nature has devised an impressive array of proteins that regulate Ca 2+ transport across cell membranes (Berridge et al., 2003). Neurotransmitter receptors play an essential role in this process. Metabotropic receptors increase [Ca 2+ ] i by modulating voltage-gated Ca 2+ channels or emptying internal Ca 2+ stores (Ross et al., 2005). Ionotropic receptors, like the ATP-gated P2X receptor, take a more direct approach. P2X receptors are ligand-gated cation channels that increase [Ca 2+ ] i by forming a cationpermeable pore (Benham and Tsien, 1987). They are expressed in a diverse range of tissues and are particularly abundant in the nervous system (Khakh, 2001;Illes and Ribeiro, 2004;Khakh and North, 2006). Seven full-length subtypes (P2X 1-7 ) are expressed in mammals, and most are capable of forming both homo-and hetero meric receptors (Torres et al., 1999;North, 2002). The percentage of the ATP-gated current attributed to Ca 2+ (Pf%) varies from 3ف to 15% depending on the subunit composition of the oligomeric receptor and the species of origin (Egan and Khakh, 2004). It is large enough to stimulate transmitter release from neurons (Pankratov et al., 2006), hormone release from endocrine glands (Troadec et al., 1998), and contraction of vascular smooth muscle (Ramme et al., 1987;Gitterman and Evans, 2001) and vas deferens (Brain et al., 2002).
Identifying the functional domains responsible for regulating Ca 2+ fl ux is complicated by the lack of a clear picture of channel architecture. Each receptor is an oligomer of three subunits, with individual subunits comprised of intracellular amino and carboxy termini linked by two transmembrane domains and an extracellular loop (North, 2002). Mutagenesis studies indicate a role for the pore-lining second transmembrane segment (TM2) in the cation permeability (Migita et al., 2001) and Ca 2+ current (Egan and Khakh, 2004) of at least one family member, the P2X 2 receptor. The fi rst transmembrane segment (TM1) also lines the pore, and mutagenesis of this domain is known to alter permeability and gating .
We previously reported that %6ف of the total current through the P2X 2 receptor is carried by Ca 2+ (Egan and Khakh, 2004), and that the ability of this receptor to select amongst cations involves a Ca 2+ -sensing Correspondence to Terrance Egan: egantm@slu.edu Abbreviations used in this paper: BU, bead unit; HEK, human embryonic kidney; Pf%, fractional Ca 2+ current; TM, transmembrane. domain made of three polar amino acids of TM2 (Migita et al., 2001). This domain is absent in the two P2X receptors (P2X 1 and P2X 4 ) that display higher Pf%s of 11-15%, suggesting that distinct loci underlie the divergent Ca 2+ currents of different family members. The aim of the present study was to identify the site(s) responsible for the elevated Ca 2+ current of the P2X 1 and P2X 4 receptors.

Molecular Biology and Cell Culture
We used wild-type rat P2X 3 , human P2X 4 (hP2X 4 ), human P2X 5 (hP2X 5 ), and zebrafi sh P2X 4.1 (zP2X 4.1 ) receptors, and C-terminal epitope-tagged rat P2X 1 and P2X 2 receptors (P2X 1 -EYMPME, P2X 2 -DYKDDDDK) that were made and expressed using conventional techniques. Addition of epitope did not change the EC 50 for ATP, the kinetics of the ATP-gated response (Torres et al., 1999), or, most importantly, the contribution of Ca 2+ fl ux to total current through the channel pore of either parent receptor (Table I). Point mutations were introduced with the Quikchange II site-directed mutagenesis kit (Stratagene) and verifi ed by automated DNA sequencing (Retrogen, Inc.). Chimerae were made as previously described (Haines et al., 2001a). All constructs were coexpressed with eGFP in human embryonic kidney-293 cells (HEK-293 or HEK-293T cells; American Type Culture Collection) using Effectene (QIAGEN) or Lipofectamine (Invitrogen) according to the manufacturers protocols. Transfected cells were maintained in Dulbecco's modifi ed Eagle's medium (Invitrogen) supplemented with 10% FBS (Invitrogen or HyClone), 2 mM glutamine, 50 U/ml penicillin G, and 50 μg/ml streptomycin (Invitrogen), and incubated for 24-48 h at 37°C in a humidifi ed, 5% CO 2 atmosphere. They were subsequently replated at low density onto poly-l-lysine-coated glass coverslips (Gold Seal; Becton Dickinson) the night before the experiment.

Patch-Clamp Photometry
Our use of the dye-overload method (Schneggenburger et al., 1993) is described in detail in Egan and Khakh (2004). In brief, we measured fractional calcium current (Pf%) by simultaneously measuring total membrane current and fl uorescence in cells loaded with a high concentration (2 mM) of the calcium-sensitive dye, K 5 fura-2 (for examples, see Fig. 2 A). Cells on coverslips were placed in a recording chamber mounted on the stage of a TE3000 epifl uorescence microscope (Nikon Instruments). ATP-gated current was recorded from single cells held at −60 mV using borosilicate glass recording electrodes (1B150F, World Precision Instruments), an Axopatch 200B amplifi er (Molecular Devices), ITC-16 data acquisition hardware (Instrutech), IGOR Pro software (Wavemetrics), and a G4 computer (Apple Computers). Electrodes had open-tip resistances of 1-3 MΩ and contained an intracellular solution of the following composition (in mM): 140 CsCl, 10 tetraethylammonium Cl, 10 HEPES, 2 K 5 fura-2 (Molecular Probes/Invitrogen), 4.8 CsOH, pH 7.35. Light emitted from the 100-W xenon lamp was passed through a 380-nm bandpass fi lter and directed through the microscope objective (HMC 40X ELWD Plan Fluor; Modulation Optics, Inc.) into the recording chamber by refl ection from the surface of a 400-nm dichroic long pass mirror. Light emitted by fura-2 was gathered by the objective, passed through the dichroic mirror and a 510-nm bandpass fi lter, and measured by a Model 714 Photomultiplier Detection System (Photo Technology International). We controlled the day-to-day variation in the sensitivity of the microscope/PMT by normalizing the fura-2 signal to a "bead unit" (BU). One BU equaled the average fl uorescence of seven Carboxy Bright Blue 4.6-μm microspheres (Polysciences) measured one at a time on the morning of that day's experiment (Schneggenburger et al., 1993;Frings et al., 2000). Subsequent measurements of ATP-evoked changes in the fura-2 fl uorescence of living cells were recorded as multiples of the daily BU. Cells were loaded with fura-2 by passive diffusion through the tip of the recording electrode. The time course of diffusion was measured in a subset of cells by monitoring the increase in intracellular fl uorescence that results from fura-2 entry (Pusch and Neher, 1988). The time constant of loading was 144 ± 16 s (n = 30), and a near steady-state concentration of intracellular fura-2 was reached within 01-9ف min of patch disruption. These measurements suggest that 10 min is the minimum time needed to equilibrate the intracellular compartment with the contents of the pipette, and we waited at least this long before acquiring data. The extracellular bath solution was (in mM) 140 NaCl, 2 CaCl 2 , 1 MgCl 2 , 10 glucose, and 10 HEPES, titrated to pH 7.4 with 4 NaOH. HEPES was replaced by MEPS in experiments performed at pH 5. ATP was applied for 0.2-2.0 s once every 2-3 min using triple-barreled theta glass and a Perfusion Fast-Step System SF-77 (Warner Instruments). For each construct and experimental algorithm, we used a concentration of ATP that evoked an easily measured decrease in fl uorescence of fura-2 excited at 380 nm (∆F 380 ) but avoided the problem of dye saturation ( Table I).
As previously reported, we saw no effect of short applications of submaximal concentrations of ATP on the fura-2 fl uorescence of mock-transfected cells using the methods described above (Egan and Khakh, 2004).
The Pf% was calculated as follows: Q T is total charge and equal to the integral of the leak-subtracted ATP-gated transmembrane current. Q Ca is the part of Q T carried by Ca 2+ , and is equal to ∆F 380 divided by the calibration factor F max . F max was determined in a separate set of experiments as previously described (Egan and Khakh, 2004) and equaled 0.0185 ± 0.002 BU/nC (n = 12).

Ca 2+ and Cl − Permeability Measurements
We measured the relative permeabilities of Ca 2+ and Cl − to Cs + (P Ca /P Cs and P Cl /P Cs , respectively) using a reversal potentialbased method and the Goldman equations (Hille, 2004). Membrane current was recorded using an AxoPatch 200B amplifi er, indifferent electrodes suspended in 3 M KCl agar bridges in contact with the bath solution, and the broken-patch confi guration of the whole-cell voltage-clamp technique. The solution in the recording pipette was (in mM) 150 CsCl, 10 EGTA, and 10 HEPES, brought to pH 7.4 with CsOH. Open tip electrode resistances measured 0.7-2.0 MΩ. Giga-ohm seals were established in a standard bath solution of (in mM) 150 NaCl, 1 CaCl 2 , 1 MgCl 2 , 10 glucose, and 10 HEPES, pH 7.4 with NaOH, and subsequently switched to test solutions of different ionic compositions. To measure P Ca /P Cs , the bath solution was fi rst changed to one that contained predominately CsCl (in mM: 150 CsCl, 0.1 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES), followed by one that contained predominately Ca 2+ (in mM: 112 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES). We included 0.1 mM Ca 2+ in the CsCl solution to retard pore dilation because this process causes a time-dependent change in cation permeability that could alter P Ca /P Cs (Khakh et al., 1999;Chaumont and Khakh, 2006); in theory, addition of 0.1 mM Ca 2+ to a solution of 150 mM CsCl solution is expected to have a negligible effect on the reversal potential of ATP-gated current (Lewis, 1979). We changed the membrane voltage of cells bathed in each solution from −80 to 60 mV at a constant rate (1.4 V/s) before and during applications of ATP, and measured the membrane voltage corresponding to the zero-current level (i.e., the E rev ) from the leak-subtracted currents. We calculated P Ca /P Cs as where a Cs and a Ca are the activity coeffi cients of Cs + (0.75) and Ca 2+ (0.25), respectively, ∆E rev is E rev,Ca − E rev,Cs , T is temperature (295.15°K, equal to 22°C), and F and R are universal constants. We used bath solutions containing different concentrations of CsCl to measure P Cl /P Cs . The fi rst solution had the same concentrations (154 mM) of Cs + and Cl − as the pipette solution. We applied a voltage ramp to obtain E rev of the ATP-gated current and calculated the junctional offset as the deviation of the measured E rev from the expected zero current level of 0 mV. This was then P2X 1 -EE and P2X 2 -FLAG are the epitope-tagged receptors (P2X 1 -EYMPME and P2X 2 -DYKDDDDK, respectively) that served as templates for mutagenesis. The concentrations of agonist ([ATP]) used in the study were chosen to give measurable responses while avoiding dye saturation, and the pA/pFs are the average current densities measured at these concentrations. Wild-type (WT) P2X 1 and epitope-tagged P2X 1 -EE receptors did not differ in Pf%s, and the pooled data were used as control in subsequent statistical evaluations. Although the Pf% of the tagged P2X 2 -FLAG receptor measured here is the same as that of the WT P2X 2 receptor measured in our previous work reported elsewhere (Egan and Khakh, 2004), we did not pool these two datasets because they were obtained in separate studies performed at distinct locations (Cambridge, UK versus St. Louis, MO). Thus, all comparisons in the present study refl ect differences from the P2X 2 -FLAG receptor. In the three rightmost columns, we compare the Pf%s of mutants and chimerae to the parent receptors. Data that are signifi cantly different from P2X 1 , P2X 2 , and hP2X 4 receptors are marked "Yes" and data that show no difference are marked "No." All data were obtained at extracellular pH 7.4 except where noted. a Not different from the WT P2X 1 receptor. b Unpublished data (n = 10); Li, personal communication. c Data (n = 18) from Egan and Khakh (2004 subtracted from the E rev obtained in a "low" CsCl bath solution that contained (in mM) 30 CsCl, 0.1 CaCl 2 , 1 MgCl 2 , 210 sucrose, 10 glucose, and 10 HEPES. P Cl /P Cs was calculated as o is the ratio of the intracellular and extracellular ion activities (Mitchell et al., 1997). Activity coeffi cients were determined by interpolation of the tabulated data of Robinson and Stokes (1970) and equaled 0.82 and 0.72 for 30 and 150 mM CsCl, respectively.

Data Analysis
All data are presented as the mean ± SEM for the number of experiments stated in the text. Signifi cant differences amongst groups were determined using InStat (GraphPad Software) by one-way ANOVA with Tukey's post hoc, or Student's t test where appropriate. The P values of individual datasets are quoted in the text; values ≤0.05 were considered signifi cant. We used the pooled data of wild-type P2X 1 and P2X 1 -EE receptors as controls for comparisons to mutated receptors because we measured no difference in the Pf%s of these two groups (Table I).

R E S U LT S
The percentage of ATP current carried by Ca 2+ varies signifi cantly amongst members of the P2X receptor family.
In the present study, we fi rst focused on the P2X 1 and P2X 2 receptors because they transduce appreciably different Ca 2+ fl uxes. We measured Pf%s of 11.8 ± 0.5 and 6.7 ± 0.2% for the rat P2X 1 and P2X 2 receptors, respectively (Table I). These values are signifi cantly different (P < 0.001) from each other and in good agreement with previously published results (Rogers and Dani, 1995;Egan and Khakh, 2004). With this baseline established, we examined the effect of genetic manipulation of channel structure on Pf% to gain insight into the molecular basis of the variability in Ca 2+ fl ux throughout the P2X family.
To What Extent Do Individual Transmembrane Segments Infl uence Ca 2+ Flux?
Empirical data suggest that both TM1 and TM2 line the ion-conducting pore of the P2X 2 receptor . Although no such data exist for the P2X 1 receptor, ATP activates cationic currents through chimeric P2X 1 /P2X 2 proteins with swapped transmembrane domains (Werner et al., 1996;Haines et al., 2001a), and therefore it is reasonable to assume that the pores of both channels share a roughly similar design. If so, then the disparity in Ca 2+ fl ux amongst receptors might refl ect subtype-specifi c differences in the primary structures of their pore-lining domains. To determine the infl uence of individual segments, we investigated three chimeric channels in which one (P2X 2 -TM1 x1 and P2X 2 -TM2 x1 ) or both (P2X 2 -TM1/2 x1 ) of the transmembrane segments of the P2X 2 channel were replaced by equivalent stretches of the P2X 1 channel (Fig. 1, A and B). All three chimerae formed functional channels when expressed in HEK-293 cells (Fig. 1 C).
We found that swapping transmembrane segments had quantifi able effects on Pf% (Fig. 2). The chimeric channel containing the fi rst transmembrane segment of the P2X 1 channel in the P2X 2 background (P2X 2 -TM1 X1 ) had a Pf% equal to 9.3 ± 0.6%, which was signifi cantly greater (P < 0.001) than that of the wild-type P2X 2 channel. Substitution of both transmembrane segments (P2X 2 -TM1/2 X1 ) had no greater effect (Pf% of 8.9 ± 0.5%) than replacing TM1 alone. In stark contrast, the chimeric P2X 2 channel containing the TM2 of P2X 1 , (P2X 2 -TM2 x1 ) exhibited a Pf% of 2.6 ± 0.3% that was signifi cantly lower (P < 0.001) than both wild-type channels ( Table I). These results suggest either that: (1) both transmembrane domains contribute to the regulation of Ca 2+ current, each in their own way; or (2) construction of the chimerae produced nonspecifi c changes in the poreforming domains that affect Pf%. To explore these possibilities, we used the less invasive methods described below.
The Ca 2+ Flux of the P2X 1 Receptor Is pH Sensitive The considerable Ca 2+ fl uxes of many ligand-gated channels result from an interaction with negatively charged acidic amino acids in the mouth of the pore (Keramidas et al., 2004;Jensen et al., 2005). We looked for an effect of ionized side chains by measuring the Pf% of P2X 1 and P2X 2 receptors at a lower pH where a greater proportion of the COO − side chains would be protonated and hence neutral (Hille et al., 1975;Green and Andersen, 1991) (Fig. 3 A). The Pf% of the wild-type P2X 1 receptor at pH 5.0 was 5.8 ± 0.8%, a signifi cant (P < 0.001) reduction when compared with that measured at pH 7.4. In contrast, the Pf% of the wild-type P2X 2 receptor was unaffected by changing pH (Fig. 3 D). These data suggest that the electrostatic attraction of Ca 2+ by one or more acidic amino acids is responsible for the high Pf%s of the P2X 1 receptor, and that titratable carboxylates are unlikely to play a role in Ca 2+ fl ux through the P2X 2 receptor.
Acidic Residues Regulate the Ca 2+ Flux of the P2X 1 Receptor Next, we used site-directed mutagenesis to locate the acidic amino acid(s) responsible for the high Ca 2+ fl ux of the P2X 1 channel. The data obtained using chimeric receptors suggested that the search should fi rst center on the transmembrane domains. We discounted the conserved aspartate of TM2 because it is present in all family members (bold black residues of Fig. 1 B), and instead looked for differences in the primary sequences of the transmembrane domains of channels that show higher (P2X 1 , P2X 4 ) and lower (P2X 2 ) Ca 2+ fl uxes. P2X 1 and hP2X 4 channels have the highest Pf%s of all members of the extended family (Egan and Khakh, 2004). Both channels have acidic residues at homologous positions in TM1 (Glu 52 -P2X 1 and Glu 51 -hP2X 4 ) and TM2 (Asp 327 -P2X 1 and Asp 331 -hP2X 4 ) that are missing in the P2X 2 channel (bold red residues of Fig. 1 B).
We engineered P2X 1 mutants in which one or both of these were replaced by the amino acids that occupy the equivalent positions in the P2X 2 receptor (Gln 52 and Ser 326 ). Neutralizing fi xed charge in TM1 (P2X 1 -E52Q) signifi cantly (P < 0.001) reduced Pf% to 8.4 ± 0.4% (Fig. 3 B and Table I). Removing the fi xed charge in TM2 (P2X 1 -D327S) had no signifi cant effect (Pf% equals 10.5 ± 0.5%). However, neutralizing both charges (P2X 1 -DM) caused a greater reduction (6.1 ± 0.6%) than neutralizing the charge of either TM1 (P < 0.01) or TM2 (P < 0.001) alone, and the sum of the effects of the single mutations (42% reduction in Pf%) was roughly equal to the effect of the double mutation alone (48% reduction in Pf%). Further, lowering the pH to 5.0 signifi cantly (P < 0.01) reduced the Pf% of P2X 1 -E52Q to 6.0 ± 0.7% but had no effect on P2X 1 -DM (5.8 ± 1.6%), suggesting that the resident charge at , hP2X 4 (TM1, Gly 29 -Lys 52 ; TM2, Asp 331 -Leu 358 ), and zP2X 4 (TM1, Gly 32 -Lys 55 ; TM2, Asn 340 -Leu 367 ) receptors. The acidic amino acids that are the focus of this study (Glu 52 -P2X 1 , Glu 51 -P2X 4 , Asp 327 -P2X 1 , and Asp 331 -P2X 4 ) are marked with bold red letters. The conserved aspartate (corresponding to P2X 1 -Asp 350 ) found in all family members is marked with bold black letters. The polar residues of TM2 that regulate Ca 2+ current through the P2X 2 receptor (Migita et al., 2001) are marked with bold blue letters. Note that a polar residue occupies only one of the three corresponding positions of the P2X 1 (Ser 337 , underlined) and hP2X 4 (Ser 350 , underlined) receptors. The neighboring amino acids (His 45 and Glu 46 ) of the P2X 3 receptor are shown in bold green letters. (C) Currents recorded in the presence of ATP for the P2X 1 , P2X 2 , and the three chimeric receptors described in the text. ATP was applied for 2 s (open horizontal bars) at concentrations of 10 μM for the P2X 2 -TM1/2 X1 chimera and 3 μM for the others. 3 and 4, the sampling rate of the fl uorescence signals are reduced by decimation to 10 Hz, converted to Q Ca (where Q Ca =F 380 / (F max *BU)), normalized to peak Q T , and then multiplied by 100%. The amplitude of the steady-state value of each trace is equal to Pf%. (C) The bar graph shows the mean and standard error of the Pf% values. Values signifi cantly different from the P2X 1 and P2X 2 receptors are marked with "a" and "b," respectively. position 327 that is present in P2X 1 -E52Q but missing in P2X 1 -DM does affect Pf%. Taken together, these data argue that both Glu 52 and Asp 327 interact with Ca 2+ in the mouth of the pore, although the relative contribution of each amino acid is different.
We also performed the converse experiment of placing carboxylates at appropriate sites in the P2X 2 receptor. We generated three mutant P2X 2 receptors in which one or both of Gln 52 and Ser 326 were replaced by glutamate or aspartate, respectively (Fig. 3, C and D). The Pf% for the single mutants P2X 2 -Q52E and P2X 2 -S326D were 11.2 ± 0.8 and 11.0 ± 0.9%, respectively. Both values are significantly greater (P < 0.001) than the wild-type P2X 2 receptor and approximately equal to the wild-type P2X 1 channel. Placing both mutations in a single construct (P2X 2 -DM) did not increase Pf% more than either mutation alone. Lowering the pH to 5.0 signifi cantly (P < 0.001) reduced the Pf% of P2X 2 -DM to 6.6 ± 1.3%, (Fig.  3 A), suggesting that the increase in Pf% results from an electrostatic effect of the added charge and not an unintended change in protein structure.
The Pf% of the P2X 4 Receptor Decreases when Fixed Charge Is Removed Next, we looked at the effects of mutagenesis on the hP2X 4 receptor because it shows the highest Pf% of all P2X receptors (Egan and Khakh, 2004) and, like the rat P2X 1 receptor, has fi xed negative charge at the outer edges of its transmembrane segments. The Pf% of the wild-type hP2X4 receptor was 16.0 ± 1.0% (Fig. 4). We found that neutralizing both of the fi xed charges of the transmembrane segments by mutagenesis gave a receptor (hP2X 4 -DM) with a signifi cantly (P < 0.001) reduced Pf% of 8.6 ± 0.6%. In contrast, removing a single charge had no effect. Specifi cally, the hP2X 4 -E51Q and hP2X 4 -D332S mutants had Pf%s of 14.6 ± 1.1 and 13.0 ± 0.6%, respectively, that were no different than the wild-type receptor. In this respect, the single charge deletion mutants of the hP2X 4 receptors resemble the single charge addition mutants of the P2X 2 receptor; the presence of one charge per subunit is enough to maintain a high Ca 2+ fl ux through the pore.
As a fi nal test, we measured the Pf% of the wild-type zebrafi sh P2X 4.1 receptor (zP2X 4.1 ) (Kucenas et al., 2003). It lacks the two relevant formal charges found in hP2X 4 receptors (Fig. 1 B) and therefore naturally replicates the charge reduction of P2X 4 -DM. In keeping with our hypothesis, the Pf% of the zP2X 4.1 receptor at 7.3 ± 1.3% was signifi cantly smaller than the hP2X 4 receptor and not different from the rat P2X 2 and hP2X 4 -DM receptors (Fig. 4 B).
Neutralizing Fixed Charge Alters P Ca /P Cs but Not P Cl /P Cs A caveat to using patch-clamp photometry to draw conclusions about the Ca 2+ fl ux of cation channels is that it does not account for the possibility that some mutations may render the channels anion permeable. With the exception of the human and chick P2X 5 receptors, most wild-type P2X receptors are cation selective and therefore impermeable to anion . However, it is possible that some mutations increase Cl − permeability, and under the conditions of our experiments (equimolar [Cl − ] on either side of the membrane and a holding potential of −60 mV), such an effect would lead to an apparent inward current caused by effl ux of Cl − , a larger Q T , and a reduction in Pf% that does not refl ect a change in Ca 2+ fl ux per se. To differentiate a change in Ca 2+ permeability from a change in Cl − permeability, we acquired reversal potential data from ATP-gated currents obtained from cells bathed in a range of extracellular solutions (see Materials and methods).
Switching the bath solution from one that contained 150 mM CsCl to one that contained 112 mM CaCl 2 caused a rightward shift in the reversal potential of the ATPgated current of the wild-type P2X 1 and hP2X 4 receptors ( Fig. 5 A) that translated to P Ca /P Cs values of 3.6 ± 0.2 and 4.6 ± 0.4, respectively (Table II). The shifts were smaller for P2X 1 -DM and hP2X 4 -DM receptors that lack the relevant formal charges, and the resulting P Ca /P Cs s were signifi cantly (P < 0.001) lower than the wild-type templates at 2.6 ± 0.1 and 2.8 ± 01, respectively. They were not signifi cantly different from that of the P2X 2 receptor that measured 2.9 ± 0.1 (Table II). Again, these data support our contention that formal charge infl uences the Ca 2+ dynamics of P2X 1 and P2X 4 receptors.
In contrast, Cl − permeability was not affected by the mutagenesis (Fig. 5 B). We fi rst measured the P Cl /P Cs of the hP2X 5 receptor because it shows a signifi cant Cl − permeability (Bo et al., 2003). As expected, we found that switching to the 30 mM CsCl bath solution caused a -6فmV shift in E rev ; the calculated P Cl /P Cs was 0.66 ± 0.12 (Fig. 5 B). We then measured the Cl − permeability of wild-type and double mutant P2X 1 and P2X 4 receptors. Shifting the extracellular solution from one that contained 150 mM CsCl to one that contained 30 mM CsCl caused an approximately −30 mV shift in the ATP-gated current in all four constructs, demonstrating that both the wild-type and mutant receptors are largely impermeable to anions (Table II). These data support the hypothesis that the changes in Pf% that we measure after mutagenesis refl ect specifi c effects on Ca 2+ fl ux.

P2X 3 Has Fixed Charge but a Relatively Low Pf%
The glutamate of TM1 is conserved in only one other P2X subtype as Glu 46 of the P2X 3 receptor. However, the P2X 3 receptor exhibits the lowest Ca 2+ fl ux of all family members (Egan and Khakh, 2004). Unlike P2X 1 and P2X 4 receptors, the P2X 3 receptor contains a positively charged amino acid, His 45 , immediately upstream to the conserved Glu 46 residue (green residues of Fig. 1 B). His 45 may provide a countercharge in the pore that serves to partially shield the neighboring Glu 46 or simply repel Ca 2+ , thus attenuating the ability of the carboxylate to attract Ca 2+ . To test this hypothesis, we constructed a mutant P2X 3 receptor in which His 45 was replaced by the tyrosine found at the homologous position of the P2X 1 receptor. This produced a small but signifi cant (P < 0.05) increase in Pf% to 6.7 ± 0.5% compared with the wild-type P2X 3 control of 4.8 ± 0.3% (Table I).
We also generated mutant P2X 1 receptors in which Tyr 51 was replaced by either a basic histidine (P2X 1 -Y51H) or a neutral valine (P2X 1 -Y51V). We found that the Pf% of P2X 1 -Y51H was signifi cantly reduced (6.3 ± 0.8%) compared with the wild-type P2X 1 receptor. This decrease was unlikely to be caused by a gross disruption of channel structure because substituting neutral valine (the corresponding residue of P2X 2 ) for Tyr 51 had no effect on Pf% (Table I). Taken together, the data suggest that the identity of the amino acid just upstream of the conserved charges of P2X 1 and P2X 3 receptors helps to determine the magnitude of the Ca 2+ fl ux through the channel pore.

D I S C U S S I O N
We identifi ed acidic residues at the outer ends of the transmembrane domains that contribute to the high fractional Ca 2+ current of ATP-gated P2X 1 and P2X 4 receptors. We found that acidifi cation decreased the relatively large Ca 2+ current of the P2X 1 receptor but had no effect on the smaller Ca 2+ current of the P2X 2 receptor, suggesting that only the former uses acidic amino acids to discriminate amongst permeant cations. (We also looked at the effect of a lower pH on the hP2X 4 receptor. The concentration-response curve of the P2X 4 receptor shifts to the right at lower pHs, necessitating the use of a very high (>1 mM ATP) concentration of ATP to evoke a measurable ligand-gated current. At these high concentrations, applications of ATP evoked biphasic changes in intracellular calcium that did not follow the time course of the integrated current, making measurements of Pf% untenable.) The magnitude of the reduction is surprising when one considers the predicted degree of protonation of the COO − side chains. We expected to measure a modest reduction in Pf% upon lowering the pH to 5.0, a value close to the pK a of glutamate and aspartate in aqueous solution, and where half of the carboxyl groups are protonated and thus unable to attract Ca 2+ (Falke et al., 1994). Instead, we found that acidifi cation produced a Pf% equal to that of mutant receptors lacking both formal charges (i.e., P2X 1 -DM). These data suggest that the pK a s of Glu 52 and Asp 327 of the P2X 1 receptor may be signifi cantly higher than predicted from measurements of pure aqueous solutions of amino acids, an idea in keeping with published reports that the local microenvironment of a protein greatly infl uences the degree of ionization of its constituent amino acids (Fersht, 1985;Klockner et al., 1996;Seifert et al., 1999;Petsko and Ringe, 2004). Of particular interest is the positive charge provided by a conserved lysine (e.g., Lys 53 of the P2X 1 receptor) that sits immediately downstream of Glu 52 . Placing positive charge close to an acidic amino acid is enough to shift the latter's pK a to 7.0 or higher (Falke et al., 1994). We have not pursued a study of this lysine because it is fully conserved and therefore unlikely to play a role in determining the dissimilar Ca 2+ fl uxes measured across the family. However, its role in the absolute magnitude of the Ca 2+ fl ux should be considered in future experiments.
The idea that individual P2X receptors use distinct domains for cation selection is supported by the marked changes in Pf% observed in P2X 1 /P2X 2 chimerae. Replacing the TM1 of the less Ca 2+ -permeable P2X 2 receptor with that of the more permeable P2X 1 receptor The raw data traces show ATP-gated currents evoked in solutions that contained either Cs + (blue traces) or Ca 2+ (red traces) as the dominant extracellular cation. The change in E rev measured after switching from the Cs + to Ca 2+ solution was smaller for the double mutant receptors (right traces) than it was for their wild-type templates (left traces), suggesting that eliminating formal charge decreased P Ca /P Cs . (B) The raw data traces show ATP-gated currents evoked in extracellular solutions that contained either 150 mM (blue traces) or 30 mM CsCl (red traces). For the wild-type P2X 1 and hP2X 4 recep-tors (top and middle left graphs), the change in E rev measured upon switching to the low CsCl solution was 33-03ف mV as expected for a channel that showed a low Cl − permeability. The same was true for the double mutant receptors that lacked the relevant formal charges of acidic amino acids (right traces), showing that mutagenesis did not change P Cl /P Cs . The change in E rev was smaller in hP2X 5 receptors that show a signifi cant Cl − permeability (bottom left graph). signifi cantly enhanced Ca 2+ fl ux, as did addition of a single formal negative charge to the extracellular end of TM1 (P2X 2 -Q52E). These data are important for three reasons: fi rst, they add weight to the proposition that TM1 lines the outer segment of the ion channel (Haines et al., 2001b); second, they show for the fi rst time that TM1 makes a measurable contribution to cation selection; and third, they suggest that the divergent Ca 2+ currents of the P2X 1 and P2X 2 subtypes result in part from differences in the primary sequences of their TM1s.
The case of TM2 is more complex. We found that adding fi xed negative charge to TM2 of the P2X 2 receptor increased Pf% only when Thr 336 , Thr 339 , and Ser 340 are unperturbed (compare P2X 2 -S326D to P2X 2 -TM2 X1 ), a fi nding that supports previous reports that the polar residues of TM2 play an important role in Ca 2+ fl ux in this receptor subtype (Egan and Khakh, 2004). Two of these are replaced by neutral amino acids in the P2X 2 -TM2 X1 chimera, thus depriving the parent P2X 2 receptor of polar side chains that may provide the countercharge needed to partially dehydrate Ca 2+ at a constriction within the pore (Migita et al., 2001). If so, then P2X 1 receptors either use other means to overcome the energy barrier provided by the constriction or lack it entirely. An alternative explanation is that the decrease in Pf% in the P2X 2 -TM2 X1 chimera refl ects nonspecifi c changes in the topology of the pore brought about by swapping one TM2 with another. This latter possibility is diffi cult to ignore, and additional experiments are needed to fi rmly establish a role for polar amino acids in ion permeation in some P2X receptors.
Many classes of ligand-gated cation channels use the formal charge of acidic amino acids positioned near the pore to facilitate Ca 2+ transport into cells (Keramidas et al., 2004;Jensen et al., 2005). P2X receptors have three sites that could serve a similar function. One of these is fully conserved in TM2 (see bold black letters in Fig. 1  B) and therefore unlikely to explain the differences in Ca 2+ fl ux measured here. Two nonconserved sites are present in P2X 1 and P2X 4 receptors but not in the P2X 2 receptor, and we found that removing both charges signifi cantly decreased the Pf% and P Ca /P Cs without affecting P Cl /P Cs . Thus, our data are consistent with a model that uses the oxygen atoms of COO − side chains to form an electrostatic ring that attracts cations. The attraction is stronger for Ca 2+ than for Na + , and therefore could explain the high Ca 2+ permeability and fl ux of P2X 1 and P2X 4 receptors. The location of the ring in the permeation pathway is unknown because the inner and outer limits of the pore are poorly defi ned . We favor the hypothesis that it lies in the extracellular vestibule for the following two reasons. First, such a model is consistent with those that explain the high Ca 2+ fl uxes of other ligand-gated cation channels (Barry and Lynch, 2005). Second, models that incorporate the formal charges of aspartate and glutamate side chains into Ca 2+ binding sites within the pore often result in channels that show Ca 2+ block of monovalent current (Voets et al., 2004), and no such effect is reported for either the P2X 1 or P2X 4 receptors.
We suggest the simple hypothesis that the formal charges of both TM1 and TM2 infl uence the fl ux of Ca 2+ through P2X 1 and hP2X 4 channels in a nonadditive fashion. We found that adding charge to either transmembrane domain signifi cantly enhanced Ca 2+ fl ux of the P2X 2 receptor, whereas removing the charge of either Glu 51 or Asp 332 had only a small effect on the Pf% of the hP2X 4 receptor. The results are therefore consistent, for together they suggest that (1) a single charge is suffi cient to cause the elevated Ca 2+ permeability noted in the P2X 2 -DM and wild-type hP2X 4 receptors; and (2) there is very little summation in terms of increasing Pf% when both charges are present in the outer parts of TM1 and TM2.
For the P2X 1 receptor, it appears that Glu 52 of TM1 might play a larger role than Asp 327 of TM2. We found that removing Glu 52 signifi cantly reduced the Pf% of the P2X 1 receptor, whereas removing Asp 327 had no effect, as might be expected if Asp 327 is innocuous. Although these fi ndings seem to invalidate the dual-charge hypothesis discussed above, two experiments suggest that Asp 327 does infl uence Ca 2+ fl ux through the channel. First, subtracting both charges (i.e., P2X 1 -DM) has a signifi cantly greater effect on the Pf% than removing either Glu 52 or Asp 327 alone. Second, acidifi cation decreases the Pf% of P2X 1 -E52Q but has no effect on P2X 1 -DM, and the only difference between these two mutants is the presence of Asp 327 . It is diffi cult to explain why eliminating the glutamate of TM1 significantly decreases the Pf% of P2X 1 but not P2X 4 receptors, except to say that structural differences in TM2 may make the Asp 327 of the P2X 1 receptor either less accessible to Ca 2+ or less charged than either the Asp 332 of the hP2X 4 receptor or the Asp 326 of the P2X 2 -S326D mutant.
P2X 1 , P2X 2 , and P2X 4 receptors have remarkably different phenotypes and display unique agonist and antagonist profi les, rates of desensitization, Ca 2+ permeabilities, and traffi cking (North, 2002). If function follows structure, then structural differences between these subtypes are to be expected. Fig. 6 shows cartoon representations of P2X 1 , P2X 2 and P2X 4 receptors that incorporate two different models of Ca 2+ transport. We assume that each channel is comprised of three subunits (Nicke et al., 1998;Aschrafi et al., 2004;Barrera et al., 2005;Mio et al., 2005), and that each subunit contributes two transmembrane segments to the pore (North, 2002). In P2X 1 and P2X 4 receptors, Ca 2+ current is enhanced by a ring of charge made of glutamate and aspartate residues positioned in the outer mouth of the pore. These residues could function to accumulate extracellular Ca 2+ in the permeation pathway by a simple electrostatic attraction, or form a binding site that selects Ca 2+ over monovalent cations (Hille, 2004;Jensen et al., 2005). As mentioned above, the latter hypothesis seems doubtful because, unlike P2X 2 receptors, Ca 2+ does not block monovalent cation current through P2X 1 receptors as expected for a binding site model (Evans et al., 1996). The extracellular ring of charge is missing in the P2X 2 receptor (Fig. 6, C and D). Instead, polar residues in the middle of the pore provide the countercharge needed to dehydrate Ca 2+ in a narrow part of the pore (Migita et al., 2001). In this model, the narrow constriction provides an energy barrier that impedes current fl ow through the channel, and the relative conductance of cations is determined by their ability to momentarily "bind" to the polar side chains as they shed water. The presence of an intrapore binding site in P2X 2 receptors is supported by two fi ndings. First, ATP-gated currents saturate with increasing concentrations of extracellular Na + (Ding and Sachs, 1999b). Second, monovalent current is blocked by low millimolar concentrations of extracellular Ca 2+ (Nakazawa and Hess, 1993;Ding and Sachs, 1999a).
One question then remains: how well do these two mechanisms describe the Ca 2+ current of other family members? Fixed charge is conserved in the highly Ca 2+permeable P2X 1 and P2X 4 receptors, but is also present in the less permeable P2X 3 and P2X 7 receptors. We have not investigated P2X 7 receptors so far, but experiments with P2X 3 receptors suggest again that each subunit uses a slightly different method to select amongst cations. We suggest that the Pf% of the P2X 3 receptor is relatively low because of the presence of a unique His 45 just upstream of Glu 46 . The positive charge it supplies might serve to partially neutralize the negative charge of Glu 46 and hinder the electrostatic interaction with Ca 2+ . However, it is worth noting that neutralizing His 45 did not elevate the Pf% of the P2X 3 receptor to a value equal to that of P2X 1 receptor, suggesting that additional factors contribute to the lower Pf% measured for P2X 3 receptor. Figure 6. Schematic representations of the ion permeable pores of trimeric P2X 1 , P2X 2 , and P2X 4 receptors. Residues in TM1 are marked with black letters, and residues in TM2 are marked with red letters. The leftmost panels show side (A and E) and top (B and F) views of P2X 1 and P2X 4 receptors. The mouth of the pore is surrounded by an outer ring of six acidic amino acids (two from each of three subunits). The outer ring is missing in the P2X 2 receptor (C and D). Also shown are the three polar residues within TM2 of the P2X 2 receptor that are thought to form a polar ring at a narrow section of the channel pore. Only one of these sites is occupied by a polar amino acid in the P2X 1 and P2X 4 receptors. TM1 also contains a conserved tyrosine (Tyr 43 ) that may be properly positioned to contribute to the polar ring.


extfieldsofstudy: Chemistry Medicine

text:
Proto‐oncogene TCL1: more than just a coactivator for Akt

Serine threonine kinase Akt, also called PKB (protein kinase B), plays a central role in regulating intracellular survival. Deregulation of this Akt signaling pathway underlies various human neoplastic diseases. Recently, the proto‐oncogene TCL1 (T cell leukemia 1), with a previously unknown physiological function, was shown to interact with the Akt pleckstrin homology domain, enhancing Akt kinase activity; hence, it functions as an Akt kinase coactivator. In contrast to pathological conditions in which the TCL1 gene is highly activated in various human neoplasmic diseases, the physiological expression of TCL1 is tightly limited to early developmental cells as well as various developmental stages of immune cells. The NBRE (nerve growth factor‐responsive element) of the proximal TCL1 promoter sequences can regulate the restricted physiological expression of TCL1 in a negative feedback mechanism. Further, based on the NMR structural studies of Akt‐TCL1 protein complexes, an inhibitory peptide, “Akt‐in,” consisting of the βA strand of TCL1, has been identified and has therapeutic potential. This review article summarizes and discusses recent advances in the understanding of TCL1‐Akt functional interaction in order to clarify the biological action of the proto‐oncogene TCL1 family and the development avenues for a suppressive drug specific for Akt, a core intracellular survival regulator.—Noguchi, M., Ropars, V., Roumestand, C., Suizu, F. Proto‐oncogene TCL1: more than just a coactivator for Akt. FASEB J. 21, 2273–2284 (2007)

The proto-oncogene TCL1 family consists of the 106, 114, and 128 amino acids-MTCP1, TCL1, and TCL1b proteins, respectively-with a predicted molecular mass of 13 (MTCP1), 14 (TCL1), and 15 kDa (TCL1b). These amino acids share a relatively high sequence homology (40% identity, 61% homology between MTCP1 and TCL1, 36% identity, 63% homology between MTCP1 and TCL1b at the amino acid levels in humans) (Fig.  1A). The NMR solution structure of human MTCP1 was elucidated first (7,8), followed by the RX structures of human TCL1 (9) and murine MTCP1 (10). Subsequently, a model was proposed for both human and murine TCL1b based on the solved structures of the other members of the family (11). Consistent with the relatively high sequence homology among the TCL1 family proteins, these proteins share a common 3-dimensional structure that consists of an orthogonal 8-stranded ␤ barrel with a unique topology (Fig. 1B). The antiparallel ␤ strands of variable length are arranged into two very similar up-and-down, 4-stranded ␤-meander motifs, connected by a long, poorly structured loop that wraps around to form the barrel. Strands ␤A, ␤B, ␤E, and ␤F form four short staves on one face of the barrel while strands ␤C, ␤D, ␤G, and ␤H form the four other long staves on the opposite face. Following the criteria given by Murzin et al. (12), this structure is classified into the category of the "filled barrel," in which the inside of the barrel is tightly packed and hydrophobic. It is interesting that MTCP1 (and possibly TCL1b) is a monomeric protein, whereas TCL1 forms a tight dimer in the crystal and in solution. An exception to the ␤ barrel structure of TCL1 proteins arises from the alternative splicing of MTCP1 transcripts, which results either in low expression of the 13 kDa TCL1 protein family or in moderate to high expression of the 8 kDa cystein-rich helical protein (13)(14)(15)(16), with mitochondrial location and yet unknown function. The unique topology of TCL1 proteins has prompted intensive studies of their folding properties. Monitoring the unfolding reaction of MTCP1 under denaturating conditions (chemical reagents, high pressure, etc.) by NMR or fluorescence spectroscopy has revealed the slowest rate of unfolding ever observed for a protein (17,18).
In yeast screening of Akt to search for an interacting Akt partner, we have demonstrated that TCL1 interacts with the pleckstrin homology (PH) domain of Akt (19). NMR studies further supported the structural basis of this interaction (20,21). This interaction was functional, since we have demonstrated that TCL1 enhances Akt kinase activity, and therefore functions as an Akt kinase coactivator (19,22). By means of mutational studies using "reversed" yeast two-hybrid screening, we demonstrated that both Akt interaction and dimerization of the TCL1 were required for complete function of TCL1 in enhancing Akt kinase activity (23).
Akt, also called PKB, was originally identified from the AKT8 acute transforming retrovirus, which causes mouse thymoma (24 -26). The activation process of Akt has been clarified recently. After growth factors are bound to cell surface receptors, Akt is translocated to the plasma membrane secondary to bind the PH domain with phosphatidylinositol (PI)-3,4 bisphosphate (PIP 2 ) and PI-3,4,5-triphosphate (PIP 3 ). A number of growth hormones activate PI3K (phosphoinositide 3-kinase), which leads to production of PIP 2 and PIP 3 at the inner leaflet of the membrane (24,27).
The activation process of Akt is regulated by phosphorylation at two regulatory sites, threonine 308/309/ 305 and serine 473/474/472 (Akt1/2/3, respectively), with phosphorylation of both required for maximum kinase activity. PDK1 (3-phosphoinositide-dependent protein kinase 1) has been identified as the primary kinase phosphorylating Akt on Thr 308. Interaction of PIP 3 with the VL1 loop of the Akt-PH domain alters the conformation, which allows PDK1 access to threonine 308/309/305 of Akt and triggers phosphorylation (28 -30). Activation of Akt is also regulated by phosphorylation events within the conserved C-terminal hydrophobic motif (30,31). Both serine 473 (Ser-473 phosphorylation and membrane anchoring are required for Thr 308 phosphorylation (32) and the complete activation of Akt. Sequence alignment of the human members of the TCL1 proto-oncogene family. Identical residues are shaded in dark blue, homologue residues in light blue. Numbering of the sequences is one of the TCL1 in humans. Elements of secondary structure are indicated on top of the sequence. The three TCL1 family proto-oncogenes consist of the 114, 106, and 128 amino acids-TCL1, MTCP1, and TCL1b proteins, respectively-with a predicted molecular mass of 14 (TCL1), 13 (MTCP1), and 15 kDa (TCL1b). TCL1 family proteins share a relatively high sequence homology (considering only human proteins: 40% identity, 61% homology between MTCP1 and TCL1, 36% identity, 63% homology between MTCP1 and TCL1b) (1)(2)(3). B) Crystal structure (ribbon diagram) of TCL1. Proto-oncogene TCL1 forms a dimer in the crystal structure as well as in solution. TCL1 proteins share a common unique topology: two symmetric 4-stranded ␤ meanders (strands ␤A to ␤D and strands ␤E to ␤H) wrap around in an 8-stranded filled ␤ barrel analogous to the structure of MTCP1. Strands A and E (shown by arrows from the top) are involved in association with Akt PH domain. Strand C (shown by arrows at the bottom) is responsible for homodimerization of the TCL1 protein. Both Akt association and homodimerization of TCL1 are required for the complete function of TCL1 to enhance Akt kinase activity (20,23,47).

THE MOLECULAR MECHANISMS OF TCL1-INDUCED AKT ACTIVATION
An essential breakthrough in understanding the function of TCL1 protein occurred when TCL1 was identified in a yeast two-hybrid search for proteins that interact with Akt. TCL1 binds to Akt and forms a hetero-oligomer within this protein complex, activating Akt (19,22).
How does TCL1 enhance Akt kinase activity? It is well documented that dimerization can lead to activation of the surface receptor kinases (e.g., VEGF-R, PDGF-R) and their intracellular responses (38 -40). Similarly, intracellular nontransmembrane kinase, ASK1, has been shown to be activated by dimerization (41). It has been reported that TEL-JAK2 fusion protein causes human leukemia as a result of oligomerization and constitutive kinase activation (42). Moreover, a conditionally activated Akt fused to the hormone binding domain of the estrogen receptor was able to stimulate PHAS-1 phosphorylation (43), suggesting that Akt dimerization can promote kinase activity. The PH domain is also thought to mediate the formation of a multimeric Akt complex associated with Akt activation (44).
To dissect the molecular mechanisms of TCL1-induced Akt activation, in vitro kinase assays were performed using chimeric Akt molecules. This process demonstrated that oligomerization of TCL1 with Akt molecules in close physical proximity facilitated Akt transphosphorylation on the regulatory Ser-473 site (and possibly Thr-308), promoting kinase activity. PDK1 (3-phosphoinositide-dependent protein kinase 1) has been identified as the primary kinase responsible for phosphorylating Akt on Thr 308 (45). It is important that TCL1 did not trigger activation of Akt kinase, since pretreatment of PI3K inhibitor (e.g., wortmannin) could compromise augmentation of Akt kinase activity. This observation indicated that some degree of Akt preactivation, presumably sensitive to wortmannin (such as association of Akt with PIP 3 and subsequent activation by PDK1), is a prerequisite for the TCL1induced Akt activation. In this scenario, in the presence of TCL1, partially phosphorylated Akt molecules bound to TCL1 to form a hetero-oligomer in which enhancement of Akt kinase activity took place via a transphosphorylation reaction (Fig. 2) (19 -21, 23, 46).
An NMR study further demonstrated that TCL1 targets the Akt-PH domain at a site that has not yet been observed in PH-protein interactions. This binding site consists of ␤4, ␤5 strands and the C-terminal helix, and is located opposite the phospholipid binding pocket (20,21). TCL1 could bind to the Akt-PH domain via ␤4, ␤5, and the C-terminal ␣-helix of the PH domain. Dimeric TCL1 cross-linking to two Akt molecules via their PH domain brought the crucial phosphorylation residues Ser-473 (and/or Thr 308) close to each other and, as a consequence, enhanced their kinase activity. Akt-PH is an electrostatically polarized molecule that adopts the same fold and topology as other PH domains, which consists of a ␤-sandwich of seven strands capped on one top by an ␣-helix [Akt1 isoform (28), Akt2 isoform (21)]. The opposite face presents three variable loops (VL1, VL2, and VL3) that form the phosphoinositide binding pocket. Akt activation is initiated by the binding of PtdIns (3, 4) P 2 (PIP 2 ), or PtdIns (3, 4, and 5) P 3 (PIP 3 ) to the Akt-PH domain, recruiting the kinase to the plasma membrane. The binding surface on MTCP1 has been identified as the highly conserved face of the barrel that comprises the four short strands (␤A, ␤B, ␤E, and ␤F). This surface is located on the opposite side of the homodimerization surface (␤C strand) of TCL1. A low-resolution structure was obtained from the SAXS data for the Akt-PH/TCL1 complex (28). With the NMR information gathered on the binding surfaces for the Akt-PH/MTCP1 complex, this allowed us to build a functional molecular model (20). In this model, TCL1 cross-links two Akt molecules at the inner leaflet of the plasma membrane in a preactivated conformation without disrupting other PH-ligand interactions. The model was further supported by the observation that Akt-TCL1 complexes were preferentially present in the fraction near the plasma membrane (47). Thus, it is possible that this interaction can strengthen the membrane association, promote trans-phosphorylation, hinder the deactivation of Akt, and involve Akt in a multiprotein complex, explaining the array of known effects of TCL1. The binding affinities of the Akt-PH with TCL1 and MTCP1 have been determined by fluorescent anisotropy as well as by an NMR titration experiment. The interaction between MTCP1 and Akt was ϳ100fold weaker than the TCL1-Akt interaction (K d of 400 M and 5 M, respectively) (20,21,48). Together with the lack of dimerization interface, this suggests for MTCP1 a distinct mechanism of Akt coactivation, or possibly a transforming mechanism separate from the augmentation of Akt activation. The molecular mechanisms of TCL1b for Akt kinase activation appear to be of interest as well, since TCL1b, unlike TCL1, does not have a structural interface for dimerization based on the predicted 3-dimensional structure. In accordance with the prediction of the TCL1b 3-dimensional struc-ture, our preliminary experiments suggested that TCL1b could not form a dimer in coimmunoprecipitation assays (M. Noguchi and F. Suizu, unpublished observation).
To study the biological functions of TCL1 and its family oncogene, transgenic mice were created for both MTCP1 and TCL1 in B or T cell-specific lineages. Both transgenic mice exhibited lineage-specific neoplastic diseases in the immune systems in late adulthood (49 -51). Consistent with our findings, these mice exhibited a modest increase in Akt kinase activity. Rapamycin, inhibitor of mTOR, a downstream target of the PI3K-Akt pathway, suppressed tumor formation in the TCL1 transgenic mice, supporting the notion that TCL1 enhances PI3K-Akt pathways in vivo (52). TCL1deficient female mice exhibited reduced fertility because of an impaired ability for normal cleavage and development to the morula stage. Impaired T and B cell development with decreased cellularity in the hematopoietic and lymphoid tissue (bone marrow, spleen, or thymus) is compatible with deregulation of Akt activation in vivo (53,54). Incomplete suppression of Akt kinase activity observed in the TCL1 gene target animals could be due in part to the presence of five TCL1B family proteins and MTCP1 in the mouse genome. The presence of more than five isoforms of the TCL1B family protein in mice (3) could affect the phenotypic manifestation of TCL1 knockout mice to compensate for the function of physiological TCL1 and its isoforms.

MOLECULAR TARGETS FOR TCL1-INDUCED AKT ACTIVATION
Akt kinase is a major downstream target of growth factor receptor tyrosine kinase that signals via PI3K. Accumulating evidence supports the fact that serine threonine kinase Akt plays a central role in regulating intracellular survival. The Akt substrates involved in the regulation of cell death include FKHRL; the proapoptotic Bcl-2 family member Bad; glycogen synthase kinase (GSK), which stimulates glycogen synthesis; and cyclic AMP response element binding protein (CREB) (26,36,55,56).
In addition to its antiapoptotic effects, Akt plays multiple roles in regulating cellular responses in various cellular environments, including glucose metabolism, cell cycle control, angiogenesis, protein synthesis, and telomerase activity (26,57). Akt phosphorylates the serine or threonine residue of the consensus motifs (RXRXXS/T) (58,59). In proteomic approaches searching for a putative molecular target of Akt within the entire human genome, Ͼ 1000 molecules that bear the consensus Akt-phosphorylation motifs (RXRXXS/T) have been identified. 14 -3-3 protein is an adaptor protein known to interact with serine residues within the R-S-X-S/T-X-P motifs in an Akt-dependent manner (60,61). Since these two motifs often overlap each other, we searched the entire genome for molecular targets of Akt that bear the double consensus motifs (RXRSXS/T-X-P) of Akt phosphorylation (RXRXXS/ Figure 2. Model of TCL1-dependent Akt kinase activation. The proto-oncogene TCL1 functions as a coactivator for Akt. TCL1 binds to the Akt PH domain and facilitates the formation of Akt/TCL1 hetero-oligomers at the inner plasma membrane leaflet through interaction of the PH domain of the kinase with PIP 3 . The formation of the hetero-dimer brings the Akt molecules to a close physical proximity, which, as a consequence, facilitates Akt transphosphorylation on the regulatory Ser-473 (and/or Thr-308) site, promoting kinase activity and its downstream cell survival signals (19 -21, 23, 46). This mechanism can explain some of the manifestations of the human T-PLL in which TCL1 gene is up-regulated secondary to chromosomal translocations, but also promotes new insight into the molecular process of activation of Akt, the core antiapoptotic regulatory molecule.
T), as well as 14 -3-3 binding motifs (R-S-X-S/T-X-P) (Fig. 3). Among the putative Akt-targeting molecules identified, there were a number of kinases, phosphatases, and RNA binding proteins, including RNA splicing factors. Without full experimental verification of all the putative intrinsic factors, actual in vivo physiological targets of Akt in human cancer in the PI3K-Akt pathway will be a challenge. However, through these molecular targets Akt can provide cellular survival in vivo.
In a normal cellular environment, Akt is known to be activated through several growth factors, including PDGF and/or cytokines, via PI3K activation (24,26). In contrast, the expression of TCL1 family oncogenes in physiological conditions is restricted to early developmental cells as well as the later stage immune cells such as splenic mantel zone B cells (1,53,62). In early developmental stages, these external signal transduction systems for Akt activation-including surface receptors, secondary messengers, and/or intracellular signaling molecules-may not be adequately developed to provide sufficient intracellular survival signals to mediate Akt activation in vivo. Therefore, it is plausible that, through these molecular targets, endogenous expression of TCL1, which then activates Akt, may provide intrinsic survival signals in the absence of external stimulation for Akt activation in various cellular environments. Hence, deregulation of the TCL1-Akt signaling pathway can result in human cancers via a diverse array of mechanisms.

DIVERGED FUNCTION OF THREE AKT AND THREE TCL1 ISOFORMS
Three isoforms of Akt have been identified: Akt1, Akt2, and Akt3 (also called PKB␣, PKB␤, and PKB␥, respectively). These three isoforms bear highly conserved catalytic and regulatory domains, both of which are required for complete activation of Akt. In physiological situations, Akt1 and Akt2 seem to be ubiquitously expressed whereas the expression of Akt3 is more restricted, with predominance toward the heart, kidney, brain, testes, lung, and skeletal muscle (63)(64)(65). Therefore, it is of interest that although both Akt1 and Figure 3. Putative molecular targets of TCL1induced Akt activation. Activated Akt phosphorylates serine/threonine residues on the intracellular targets within the consensus motifs (RXRXXS/T) to regulate cellular responses (58,59). Recent proteomic analysis allowed us to identify the molecules that contain double consensus motifs of Akt phosphorylation (RXRXXS/T) and 14 -3-3 binding motifs (RSXS/TXP) within the entire human genome (60,61). Putative target molecules that contain double consensus motifs of both Akt phosphorylation and 14 -3-3 binding motifs (RXRSXS/T-X-P) are listed based on the analysis using PROSITE (http://au. expasy.org/prosite/). Akt2 express essentially ubiquitously in normal human tissues, expression patterns in human cancer seem to be different between Akt1 and Akt2. In human cancers, Akt1, originally identified as a gene amplified in human gastric cancer, also displays increased activity in primary carcinoma of the prostate, breast, and ovary, with poor prognosis. In contrast, Akt2 is most frequently activated in a wide variety of human cancers including pancreatic, breast, and ovarian cancers, with poor prognosis. Consistent with restricted physiological expression of Akt3, activation of Akt3 in human cancers appears to be less common, the only exception being androgeninsensitive prostate cancer, progressive melanomas, or estrogen-receptor-negative breast cancer. Overexpression of wild-type Akt2, but not Akt1 or Akt3, could transform NIH 3T3 cells, inducing invasion and metastasis in human breast and ovarian cancer. To further support the distinct role of the activation of the three Akt isoforms, each Akt isoform was recently shown to play distinct biological functions. Akt1 and Akt2 have been shown to exhibit two opposing functions in cancer cell motility and invasion through the transcription factor NFAT, a vital process underlying cancer metastasis and/or progression (66 -69). However, since both Akt1 and Akt2 are expressed ubiquitously in human tissues yet bear opposing functions, the in vivo action for transforming human cancers can be complex.
Gene-targeting mice also provided evidence that each Akt isoform has distinct biological actions in vivo. Akt1-deficient mice were viable but displayed impairment in organ growth, suggesting that Akt1 plays a dominant role in regulating cell survival. Mice deficient in Akt2 exhibited an impaired ability for insulin to act in skeletal muscle and/or liver. This is somewhat surprising as both Akt1 and Akt2 are ubiquitously expressed in mammalian tissues. It is important that the substitution of Akt1 and Akt3 did not compensate for the loss of Akt2. Therefore, a malfunction of glucose metabolism observed in Akt2, but not in Akt1, knockout mice could be due in part to malfunction of glucose metabolism in the liver, which could affect the overall deregulation of glucose metabolism in vivo. In contrast-and consistent with restricted expression of Akt3 in neurological tissues-Akt3-deficient mice exhibited a reduced brain size, affecting all major brain regions. The presence of other isoforms of Akt did not compensate for the defective phenotype of the gene-targeting animals, further supporting the distinct function of three isoforms of Akt in vivo (70). Since Akt1 and Akt2 are essentially expressed ubiquitously, the in vivo phenotype of gene-targeting animals may not be explained by the differential expression of Akt isoforms, but rather the distinct function of each Akt isoform (Akt1, Akt2, or Akt3). Given the highly conserved structure of the three Akt isoforms, it remains unclear how these divergent functions among the Akt isoforms are mediated. Since the C-terminal regions between each of these three Akt isoforms are relatively diverse (73-84%) compared with the kinase domain (90 -95%), the C-terminal regions may represent functional differ-ences among Akt1, Akt2, and Akt3. Nonetheless, the findings together could explain some of the divergent functions of Akt isoforms underlying the pathogenesis and clinical manifestations observed in human cancer patients.
In contrast to the Akt isoforms, there was only limited evidence demonstrating the divergent functions of TCL1 isoforms. The TCL1 protein family consists of three members-TCL1, MTCP1, and TCL1b-which do not contain any known functional motifs. Proteins of the TCL1 family share a relatively high degree of amino acid homology (30 -50%) and a unique, symmetrical ␤-barrel structure based on X-ray crystallographic analysis of TCL1 (see Fig. 1). Three TCL1 isoforms could interact with the Akt PH domain and enhance Akt kinase activity. In contrast to Akt1 and Akt2, which could bind to all members of the TCL1 family, Akt3 specifically interacted with TCL1 but not with MTCP1 or TCL1b. This specific association could determine the functional specificity of Akt kinase activation, since the presence of TCL1-but not the presence of MTCP1 or TCL1b-increased Akt3 kinase activity in in vitro kinase assays (46). The physiological expression of TCL1 is relatively restricted to early developmental cells as well as immune cells (1,11). Differential interaction of the three TCL1 isoforms with the three Akt isoforms may explain part of the divergent functions of the Akt isoforms. Since the three TCL1 family proteins appear to be expressed in different tissues (see below), it is possible that the three Akt family kinases and the TCL1 family play differential roles in vivo, underlying the pathogenesis of various human cancers. These observations also imply that each Akt isoform and/or TCL1 could be specific molecular targets for cancer therapy in various human cancers.

GENE REGULATION OF TCL1
In pathological conditions, TCL1 is overexpressed in a variety of human diseases, including EBV-infected B cell lymphoma, A-T, seminoma, dysgerminoma, and AIDS-related lymphoma (4,5,51,71). In contrast to pathological conditions, the physiological expression of TCL1 is primarily restricted to early embryonic cells, CD4 Ϫ CD8 Ϫ CD3 Ϫ thymocytes, CD34 ϩ CD19 ϩ pro-B cells through IgM-negative pre-B cells, and splenic mantel zone B cells (1,53,62). The relatively restricted physiological expression of the TCL1 family proteins suggests that the TCL1 gene is regulated at a transcriptional level (1,53). Consistently, recent studies clarified that Sp1 sites around the TCL1 transcriptional start site play a regulatory role in gene activation of the TCL1 oncogene (72). Nucleotide sequence analysis of the 5Ј-TCL1 promoter region revealed a TATA box with cis-regulatory elements for Nur77/NGFI-B, (NBRE, nerve growth factor-responsive element, CCAAG-GTCA), NF-B (73), FKHRL (Forkhead transcription factor) (60), and SP1 (72). It was striking that cis-regulatory elements of two of the Akt substrates (FKHRL and Nur77) and related molecules (IB for NF-B) were present within the proximal 5Ј-promoter of the human TCL1 gene.
Nur77 (NGFI-B, nerve growth factor-induced-B, TR3) was originally identified as an NGF-induced ligand-dependent transcriptional activator in PC12 cells (rat pheochromocytoma cells) (74). Two other subfamilies of NGFI-B transcription factors, Nurr1 (Nur-related factor 1) and NOR-1 (neuron-derived orphan receptor 1), have been identified in neuronal cells (74 -76). Studies demonstrated that Nur77 plays a pivotal role in T cell apoptosis in vivo and functions on mitochondria to control cell death (77).
Akt was recently shown to interact physically with Nurr77, phosphorylate at Ser-355 and, as a consequence, down-modulate DNA binding and transcription activation (78,79). Despite intensive study, none of the direct molecular targets of NBRE that regulate the cell death survival machinery have been identified. The molecular target of Nurr77/NGFI-B is not clear; however, one proposed target is steroid 21-hydroxylase (80), one of a group of related cytochrome P-450 enzymes that are required for steroid hormone biosynthesis.
We showed that the TCL1 gene is a direct target of Nur77 via Akt-induced phosphorylation of Nur77/ NGFI-B in vivo (81). To the best of our knowledge, TCL1-NBRE is the first direct target of Nur77 involving the regulation of intracellular cell death survival. The recognition sequences of NGFI-B orphan receptor superfamily transcription factors present within the 5Ј proximal TCL1 promoter were well conserved (underlined) in humans (CCAAGGTCA, Ϫ396 to Ϫ388 from ATG), mice (ACCTGGTCA, Ϫ336 to Ϫ328), and rats (ACGAGGTCA, Ϫ402 to Ϫ394) (82, 83) (Fig. 4).
Since both TCL1 and Nur77 are expressed in early developmental cells of the immune cells, the Akt-Nur77-TCL1 regulatory loop may play a role in providing survival signals during the process of thymic selection via mediation of Akt activation. In this scenario, TCL1 may play a dominant role in enhancing Akt kinase activity in order to maintain cellular survival signals in early developmental cells in vivo.
How could this negative regulatory mechanism play a role in the pathological condition of human leukemia? It is plausible that disruption of the negative feedback regulation of the TCL1 gene underlies the molecular mechanisms of human leukemia. SupT11 cells are a T cell leukemia cell line in which the TCL1 gene is activated by juxtaposition to the T cell receptor ␣ or ␤ loci by chromosomal translocations (1). Indeed, we demonstrated that PDGF stimulation did not inhibit TCL1 gene induction in SupT 11 cells. This observation suggested that PDGF-induced Akt activation could suppress TCL1 transactivation under a physiological condition in which the TCL1 gene was driving under an authentic TCL1 promoter that contains NBRE, but not in a pathological condition in which the TCL1 gene expression was primarily driven by a TCR (T cell receptor) promoter after chromosomal translocation. Loss of this negative feedback mechanism observed in SupT11 cells could contribute to constitutive activation of Akt in supT11 cells (M. Noguchi and F. Suizu, unpublished results).
The TCL1b gene, the third member of the TCL1 family of oncogenes, is located adjacent to the TCL1 oncogene on human chromosome 14q32. Both TCL1 and TCL1b are highly expressed at early developmental stages in several fetal tissues, including thymus, kidney, lung (TCL1), spleen (TCL1b), and placenta (TCL1b). Both TCL1b and TCL1 mRNAs are abundant in oocytes and two-cell embryos, but are rare in various adult tissues and in lymphoid cell lines in mice (1,11,50). Consistent with the differential expression pattern observed in TCL1 and TCL1b, 5Ј-promoter sequence of TCL1b contains no TATA box or GC-rich sequences with YY1 and c-myc, which are typical for a housekeeping gene. In humans, a TCL1b pseudogene is also present on human chromosome 5. However, only one of each mature TCL1 family protein is expressed at a transcriptional and translational level. In the mouse  (60,61), and SP1 (72). Nur77/NGFI-B, an orphan receptor superfamily transcription factor implicated in T cell apoptosis, is a substrate for Akt. TCL1 induces Akt phosphorylation, prevents the interaction of Nur77 with NBRE, and in turn suppresses TCL1 gene expression in PC12 cells in vivo (78,79). TCL1-NBRE is the first direct target of Nur77 involving the regulation of intracellular cell death survival (81). It is possible that this novel regulatory mechanism may play a regulatory role in early embryogenesis and/or immunological cells in vivo. genome, five TCL1b genes are present and coexpressed. Analogous to the structure of the TCL1b gene, the 5Ј promoter sequence of human MTCP1 located on the X chromosome bears no TATA box and is GC-rich, typical for a housekeeping-type promoter. In contrast to TCL1, the physiological expression of MTCP1, the third member of the TCL1 family of oncogenes, remains unclear. A recent study, however, suggested that SEB treatment induced MTCP gene expression in murine T cells in vivo (84). Taken together, these TCL1 isoforms are differentially expressed and, as a consequence, regulate various cellular responses. How these differentially expressed TCL1 isoforms can regulate various cellular responses in vivo remains an open question.

AKT PATHWAY AS A POTENTIAL THERAPEUTIC TARGET FOR CANCER THERAPY
Kinase activities are tightly regulated in cells, and the modes of regulation are diverse and overlapping for many disease states (40). Therefore, kinases represent important therapeutic targets, and significant resources have been invested to identify therapeutically useful kinase inhibitors.
One striking discovery was that Akt is a downstream target of PI3K, a mastermind of intracellular signaling for multiple growth factors (24,26). PI3K is a major signaling factor that catalyzes production of the second messenger PIP 3 at the cellular membrane. Since the PI3K-Akt pathway is involved in various human neoplastic diseases, Akt represents an attractive target for drug development against cancer (27,85,86) (Fig. 5). In pursuit of this aim, the binding site of TCL1 on the PH domain surface may present some interesting features. Several kinase inhibitors have been developed to target the active sites of the proteins, but the relatively conserved biochemical feature of this site often exhibited unwanted cross-inhibition among the different kinases (87,88). Analogues of PtdIns-P head groups showed limited effect despite blocking membrane recruitment of the kinase by competing for the binding of inositol-P in the binding pocket of the PH domain (89 -91). Indeed, 252 PH domains have been detected in protein sequences from an inspection of the human genome Figure 5. Targeting PI3K-Akt pathways for anti-cancer therapy. The PI3K signaling pathway is crucial to many aspects of cell growth and survival (second row from the left, major cellular function). The PI3K-Akt pathway regulates tumor suppressor genes and/or oncogenes that are structurally and/or functionally altered at the DNA level (schematic at the left). Deregulated activation of PI3K-Akt survival pathways contributes to the pathogenesis of a wide variety of human malignancies including ovarian, breast, and colon cancers (middle row, abnormal findings in cancer). Some inhibitory molecule targets for PI3K-Akt were previously developed. Shown on the right row are compounds that have been developed to control the PI3K-Akt pathway for therapeutic purposes for human cancers (right row, targeting pharmacological compounds). A synthesized phosphatidylinositol analog inhibited Akt by competing with phosphatidyl inositol. The plant-derived pigment curcuin reduced Akt activity, resulting in arrested cell growth in several prostate cancer cell lines. A compound synthesized from rotenone (degeulin) was a potential Akt inhibitor in malignant bronchial epithelial cells. API-2 (a nucleoside analog), also known as triciribin, is developed to suppress Akt kinase activity. However, hyperglycemia and hepato-toxicicity occurred as adverse effects in a human cancer trial. Pyridine derivatives, orally available alkylphospholipid, inhibited Akt phosphorylation, hence inducing apoptosis in cancer lines. Perifosine was also tested in a phase II trial in breast cancer. However, because of adverse effects, including severe gastrointestinal symptoms, not all the reagents listed are currently under clinical investigation (27, 33, 85, 86, 95-100). (92), among which Ͼ100 kinases use a PH domain for their translocation to the plasma membrane and/or subsequent activation. The PH domain family appears to be a very large family of structurally homologous proteins, but shares only moderate to low sequence similarity (93,94). The binding of PtdIns-P phospholipids is thought to be one of the common functions shared by most PH domains. In addition, PH domain can also engage specific protein-protein interactions, and the specificity of these interactions relies heavily on the specific and different nature of their surfaces. Thus, targeting a protein-protein binding surface on a kinase PH domain should yield specific inhibitors with minimum cross-reaction features.
By means of reversed yeast two-hybrid screening in combination with a random amino acid library, we have identified the ␤A and ␤E sheet of TCL1 as the Akt binding domain (23). Structural studies of Akt-TCL1 complexes further defined the physical interaction of the protein complexes at amino acid levels (20). In an attempt to develop Akt-specific inhibitors, we have generated a peptide spanning the Akt binding sequences of TCL1, named "Akt-in" (Akt inhibitor, NH 2 -AVTDHPDRLWAWEKF-COOH), which still interacted with Akt but lacked the ability for oligomerization. Interaction of Akt-in with the Akt PH domain prevented phosphoinositide binding, and hence inhibited membrane translocation and activation of Akt. As revealed by NMR chemical mapping, the Akt-in peptide bound the Akt-PH in a surface similar to that recognized by TCL1 proteins. Akt-in inhibited not only cellular proliferation and antiapoptosis in vitro, but also in vivo tumor growth (48). To support the efficacy for human cancer therapy further, in vitro proliferations were examined using human cancer cell lines for lung and ovarian cancers. Although there were some variations in their efficacies, in principle, 12 of 12 human cancer cell lines (lung or ovarian cancers) exhibited effective inhibition of proliferation in vitro (supplemental data).
Since NMR studies detected slight conformational changes on the VL1 loop upon peptide binding, we first postulated that Akt-in prevented phosphoinositide binding, thus inhibiting membrane translocation and activation of Akt. In more recent studies, NMR titration experiments revealed only a slight decrease in the affinity of Akt-PH for PIP 4 (the head group of PIP 3 ) when adding saturating concentrations of peptide (C. Roumestand, personal communication). Although binding conditions in the experimental systems may have affected the interactions, the observation suggested that the mode of action of the peptide could be more complex, and remains to be unraveled.
As an additional attempt to elucidate the mechanism of cancer cell growth suppression, a DNA array was utilized to search for activated or suppressed genes using Akt-in to dissect the molecular nature of suppressed tumor growth. Genes induced by Akt-in treatment were retinal pigment epithelium-specific protein, keratin-associated protein 9.9, alpha 2, 80 syalyltrans-ferase, interleukin 3, tectorin alpha, and interleukin 1 receptor type I. Several genes were inhibited by treatment with Akt-in, including TBX, SPANX family member, V-fos (osteosarcome viral oncogene homologue), Ets variant gene 5, Myos, heavy polypeptide, and growth hormone-releasing hormone receptor. It is likely that, through the activation and inactivation of these target genes, Akt-in could exhibit tumor cell suppression observed in vivo and in vitro. We also tried to modify the design of the inhibitory peptides that could interact with Akt-PH domains based on the corresponding amino acid sequences of the others members of the TCL1 family { [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] in TCL1B: 5-19 (GAPPDHLWVHQEG) in MTCP1]} (1-3). However, our preliminary experiments suggested that neither TCL1B nor MTCP1 peptides showed significant affinity to the Akt-PH domain. Therefore, to obtain more efficient Akt inhibitors, further dynamic modification of the design of inhibitory molecules will be required. For this purpose, the combined use of SAR (structure/activity relationship) methodology and of chemical fragment libraries to develop chemical compounds to manipulate the suppressive function of Akt-in might help to design more potent Akt inhibitors with minimal off-target activities for therapeutic use.

PERSPECTIVES
More than a decade has passed since the discovery and identification of the TCL1 family proto-oncogene. In the past several years of research, the physiological function and structures of TCL1 and its isoforms have been clarified. While TCL1 and TCL1b are coexpressed in human T-PLL as a result of chromosomal translocation, it remains unclear why only a prolymphocytic population of the T cell development could result in malignant transformation. In human T-PLL, both the TCL1 and TCL1b genes are activated by juxtaposition onto the T cell receptor ␣ or ␤ loci. Therefore, it remains unclear whether TCL1b itself (independent from TCL1) bears oncogenicity. Since TCL1b lacks a dimerization domain, the nature of TCL1b-induced Akt activation is intriguing. Structural studies may answer this question. The physiological function of TCL1 and its family of proteins in the early developmental cells in the immunological compartment needs to be determined. Although it is already known that activated Akt can translocalize to the nucleus, whether or not TCL1 is indispensable for nuclear translocation of Akt needs to be clarified. It is plausible there are additional functional interacting molecules for TCL1 family proteins that have not yet been identified.
Studies are under way to solve these issues in order to understand more fully all aspects of the biological function of TCL1 and its family of oncogenes underlying various human cancer pathogenesis.
We thank T. Yamori (Japan Cancer Institute) for the proliferation assays of human cancer cells, J. Chiorini (NIH) for critically reading the manuscript, K. Kinowaki and M. Narita (Division of Cancer Biology, Hokkaido University) for their technical assistance, and A. Kojima for secretarial assistance. M.N. is supported by grant aid from the Japanese Ministry of Education. F.S. is supported by the Uehara Memorial Foundation. C.R. and V.R. are grateful to the Association pour la Recherche sur le Cancer for a research grant. V.R. is supported by the "Ligue Nationale contre le Cancer."