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mTOR·RICTOR Is the Ser473 Kinase for Akt/Protein Kinase B in 3T3-L1 Adipocytes*

The insulin-signaling pathway leading to the activation of Akt/protein kinase B has been well characterized except for a single step, the phosphorylation of Akt at Ser-473. Double-stranded DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) gene product, integrin-linked kinase (ILK), protein kinase Cα (PKCα), and mammalian target of rapamycin (mTOR), when complexed to rapamycin-insensitive companion of mTOR (RICTOR), have all been identified as playing a critical role in Akt Ser-473 phosphorylation. However, the apparently disparate results reported in these studies are difficult to evaluate, given that different stimuli and cell types were examined and that all of the candidate proteins have never been systematically studied in a single system. Additionally, none of these studies were performed in a classical insulin-responsive cell type or tissue such as muscle or fat. We therefore examined each of these candidates in 3T3-L1 adipocytes. In vitro kinase assays, using different subcellular fractions of 3T3-L1 adipocytes, revealed that phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 phosphorylation correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKCα. PKCα was completely absent from compartments with Ser-473 phosphorylation activity. Although purified DNA-PK could phosphorylate a peptide derived from Akt that contains amino acid Ser-473, it could not phosphorylate full-length Akt2. Vesicles immunoprecipitated from low density microsomes using antibodies directed against mTOR or RICTOR had phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 activity that was sensitive to wortmannin but not staurosporine. In contrast, immunopurified low density microsome vesicles containing ILK could not phosphorylate Akt on Ser-473 in vitro. Small interference RNA knockdown of RICTOR, but not DNA-PK, ATM, or ILK, suppressed insulin-activated Ser-473 phosphorylation and, to a lesser extent, Thr-308 phosphorylation in 3T3-L1 adipocytes. Based on our cell-free kinase and small interference RNA results, we conclude that mTOR complexed to RICTOR is the Ser-473 kinase in 3T3-L1 adipocytes.

Insulin increases glucose transport in muscle and fat by stimulating the translocation of the glucose transporter isoform Glut 4 from intracellular storage pools to the cell surface (1,2), a process that requires the activation of the serine/threonine kinase Akt (protein kinase B) (3)(4)(5)(6)(7)(8).
Although the molecular events that occur after Akt activation are poorly understood, the early insulin signaling pathway has been well characterized except for one step, the phosphorylation of Akt at Ser-473. The signaling cascade is initiated when insulin binds to specific receptors at the plasma membrane (PM), 2 triggering the autophosphorylation of several critical intracellular tyrosine residues and thereby activating an intrinsic tyrosine kinase that can phosphorylate cellular substrates, most notably the insulin receptor substrate (IRS) proteins (9 -11). Tyrosine-phosphorylated IRS proteins can recruit and activate phosphoinositide 3-kinase (PI 3-kinase), which generates phosphatidylinositol 3,4,5-trisphosphate (PIP3), using inositol-containing phospholipids in the PM as substrates (12). The activity of Akt is markedly stimulated in a PI 3-kinase-dependent manner (13). This phenomenon predominantly relies on the phosphorylation of Akt on two of its amino acid residues: 1) threonine 308 in the activation loop of the kinase catalytic domain and 2) serine 473 in the "hydrophobic motif " (HM) carboxylterminal domain (14). The phosphorylations of both of these regulatory sites occur after the recruitment of Akt to the PM through the binding of its pleckstrin homology (PH) domain to PIP3 (15) and are completely ablated in vivo by the PI 3-kinase inhibitor wortmannin (16). The protein kinase responsible for phosphorylating Akt on Thr-308 is phosphoinositide-dependent kinase 1 (17)(18)(19). The identity of the kinase responsible for phosphorylating Akt on Ser-473 is controversial.
Akt is part of the protein kinase AGC (cAMP-dependent, cGMP-dependent, and protein kinase C) subfamily whose members are activated in a manner similar to Akt (20). All members possess a phosphorylation site in the activation loop equivalent to Thr-308, whereas only some require phosphorylation at the HM site. Some AGC members have a negatively charged acidic residue instead of a Ser/Thr in the C-terminal hydrophobic domain (21). Phosphorylation of the HM site or substitution with an acidic residue provides a docking site to recruit phosphoinositide-dependent kinase 1 and stimulate the phosphorylation of Thr-308 (20). X-ray crystallographic studies have suggested that in the inactive state the activation loop of Akt adopts a disordered structure that prevents the binding of ATP and protein substrates (22). Phosphorylation of Ser-473 results in a disorder to order transition, allowing interaction between the HM domain and the N-terminal lobe leading to the activation of the kinase (23).
At least 10 kinases have been proposed to function as the hydrophobic motif domain protein kinase (HMD-PK) that phosphorylates Akt on Ser-473, including integrin-linked kinase (ILK), protein kinase C␣ (PKC␣), double-stranded DNA-dependent protein kinase (DNA-PK), * This work was supported in part by National Institute of Health Grant R01 DK067229 and a research grant from the American Diabetes Association. 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. 1 To whom all correspondence should be addressed: Dept ataxia telangiectasia mutated (ATM) gene product, and the mammalian target of rapamycin (mTOR) (24). It has also been suggested that Akt can undergo autophosphorylation at Ser-473 (25); however, this is most likely not the mechanism by which insulin stimulates Ser-473 phosphorylation (15,16,26,27). Of all of the proposed candidates, evidence supporting DNA-PK and mTOR is the most compelling. Based on the observation that membrane localization is sufficient to activate Akt (15), Hemmings and co-workers used a biochemical approach to purify a constitutively active membrane kinase from HEK 293 cells capable of phosphorylating Akt on Ser-473 (28). The kinase, DNA-PK, was found to associate and co-localize with Akt at the PM as well as phosphorylate and activate Akt 10-fold in vitro (29). These results were surprising, since DNA-PK has been described as a nuclear protein involved in DNA replication, gene transcription, and DNA repair (30). Nevertheless, knockdown studies with small interference RNA (siRNA) and experiments using DNA-PK-deficient glioblastoma cells revealed that insulinstimulated Ser-473 phosphorylation was greatly impaired in both cases (29). Like DNA-PK, mTOR is a member of the family of PI 3-kinaserelated kinases whose activities are strongly inhibited by wortmannin and LY294002 (31,32). mTOR can regulate cell growth and proliferation through its activation by growth factors and nutrients (33). It is also the best characterized HM kinase, capable of phosphorylating the hydrophobic motif of p70S6 kinase when mTOR is complexed with G␤L (34) and regulatory associated protein of mTOR (RAPTOR) (35). Phosphorylation at the HM site of p70S6K (32) but not Akt (36,37) is inhibited by rapamycin. It is for this reason that mTOR was initially ruled out as being the HMD-PK for Akt. Recently, however, Sabatini and co-workers (31,38) have shown that mTOR can associate with G␤L and another protein called rapamycin-insensitive companion of mTOR (RICTOR), forming a rapamycin-insensitive complex capable of phosphorylating Akt at Ser-473. RNA interference experiments in Drosophila and human cell lines as well as several in vitro studies provide intriguing evidence that the mTOR⅐RICTOR complex can phosphorylate the HM site of Akt (31). It is still not exactly clear which if any of the proposed candidate kinases are the HMD-PK for Akt in an insulin-responsive tissue such as muscle and fat. In fact, it has been suggested that phosphorylation of Ser-473 may be due to multiple kinases that are cell type-or signaling pathway-specific (24). Several years ago, we characterized the phosphorylation of Akt at Ser-473 using a cell-free insulin-signaling assay that we developed using subcellular fractions from 3T3-L1 adipocytes (27,39). We found that insulin-stimulated Akt phosphorylation at Ser-473 was PI 3-kinase-dependent and due to a kinase localized in both low density microsomes (LDM) and a subpopulation of the PM fraction enriched in proteins associated with the actin cytoskeleton (Ext-HiP) (27). Utilizing the cell-free system as well as in vivo siRNA knockdown studies, we addressed whether ILK, PKC␣, DNA-PK, ATM, or mTOR was involved in the insulin-stimulated phosphorylation of Akt at Ser-473 in 3T3-L1 adipocytes. Our results indicate that the mTOR⅐RICTOR complex is the HMD-PK for Akt in fat cells.

EXPERIMENTAL PROCEDURES
Cell Culture of 3T3-L1 Adipocytes-3T3-L1 preadipocytes obtained from the American Type Culture Collection were grown to confluence and 48 h later subjected to differentiation as described previously (40). 3T3-L1 adipocytes were used 10 -14 days after initiating differentiation except where otherwise noted.
In Vitro Assay-Mature 3T3-L1 adipocytes were serum-starved overnight and then fractionated in the basal state as described previously, except that 1 mM dithiothreitol was included in HES (50 mM Hepes, pH 7.4, 255 mM sucrose, 1 mM EDTA, and protease inhibitors) and IC (20 mM Hepes, pH 7.4, 140 mM potassium glutamate, 5 mM NaCl, 1 mM EGTA, and protease inhibitors) buffers (39,41). Protease inhibitors (Sigma) were as described (39). Ext-HiP and salt-washed plasma membranes were isolated in the manner described previously (27). In vitro reactions were prepared by mixing various combinations of cytosol (CYT), PM, nuclear (NUC) enriched fraction, LDM, and Ext-HiP with 16 nM Akt2 (Upstate Biotechnology, Inc., Lake Placid, NY) and 10 M PIP3 (Calbiochem), prepared in a sonicated mixture of 100 M phosphatidylcholine (Avanti Polar Lipids) and 100 M phosphatidylserine (Avanti Polar Lipids). Reactions (typically 100 l, final volume) were initiated with the addition of an ATP-regenerating system (final reaction concentrations, 1 mM ATP, 8 mM creatine phosphate, 30 units/ml creatine phosphokinase, and 5 mM MgCl 2 ) and then incubated with rotation at 37°C for the indicated period of time. Assays were quenched by the addition of an equal volume of buffer A (50 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM sodium vanadate, 100 mM NaF, 10 mM sodium pyrophosphate, and protease inhibitors) containing 2% SDS.
Immunoblot Analysis-Protein samples were subjected to SDS-PAGE and transferred to nitrocellulose. Akt phosphospecific and PKC␣ (2056) antibodies were obtained from Cell Signaling Technology. DNA-PK cs (H-163) and ATM (5C2) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Two mTOR antibodies were used for immunoblot analysis, a polyclonal antibody mTOR (mTab2) from Upstate Biotechnology and a monoclonal antibody RAFT1 (611132) from BD Biosciences. RAPTOR antibodies were either from Cell Signaling Technology (4978) or from Abcam (antibody 5454). RICTOR antibodies were a kind gift from Dr. David M. Sabatini (Whitehead Institute) and were also obtained from Bethyl Laboratories. Immunoblots visualized by ECL detection (Amersham Biosciences) were quantified using NIH Image 1.62f. 6 and 12 g of control proteins were run on each gel to establish the linearity of the autoradiogram.
Immunoprecipitation of LDM-500 g of LDM in 500 l of IC buffer (with 1 mM dithiothreitol) were precleared with 40 l of protein A-agarose for 30 min. After centrifugation for 10 min at 4°C in a microcentrifuge, the supernatant was rotated overnight with 4 g of primary antibody. Samples were then centrifuged for 5 min at 4°C in a microcentrifuge to remove nonspecific aggregates that formed overnight. 30 l of protein A-agarose were added to the supernatants for 2 h at 4°C. Pellets were washed six times with IC buffer, resuspended in IC buffer, and then assayed in vitro for HMD-PK activity. ILK (3862) polyclonal antibodies (Cell Signaling Technology), mTOR (mTab1) polyclonal antibodies (Upstate Biotechnology), and RICTOR antibodies (D. M. Sabatini) were used in the immunoprecipitation studies.
HMD-PK Peptide Assay-HMD-PK activity was assayed using a modified version of Hill et al. (28). 50-l reactions were incubated for 20 min at 30°C in a buffer containing IC buffer, 1 mM dithiothreitol, 10 mM MgCl 2 , 1 M protein kinase A inhibitor peptide, 100 M ATP, 1 Ci of [␥-32 P]ATP, 0.5 mg/ml substrate (RRPHFPQFSYSASSTA), or control (RRPHFPQFAYSASSTA) peptide. Peptides were phosphorylated using 25 units DNA-PK (Promega), 0.75 mg/ml LDM, or 0.4 mg/ml Ext-HiP. 10 g/ml salmon sperm DNA was added in some cases. Reactions were quenched by adding 5 l of 100% (w/v) trichloroacetic acid and then centrifuged for 10 min in a microcentrifuge at room temperature. 35 l of the supernatant were spotted on P81 paper (Whatman), washed extensively in 0.1% phosphoric acid, and then analyzed by scintillation counting. Specific counts were calculated from the difference between the Ser-473 peptide and the control peptide.
siRNA Electroporation of 3T3-L1 Adipocytes-3T3-L1 adipocytes were electroporated according to the procedure of Jiang et al. (6). Five days after differentiation, 3T3-L1 adipocytes were detached from cell culture plates with 1 mg/ml collagenase and 0.05% trypsin, 0.02% EDTA. Cells were washed three times with ice-cold PBS and resuspended in PBS prior to electroporation. Approximately 5 million cells (half of the cells on a 150-mm dish) were resuspended in 0.5 ml of PBS, mixed with 20 nmol of siRNA duplexes, and electroporated at the setting of 0.18 kV and 950 microfarads using the Bio-Rad Gene Pulser Xcell electroporation system. Cells were immediately incubated with fresh media for 10 min before reseeding. Experiments were conducted 72 h after electroporation.

Subcellular Localizations of DNA-PK and mTOR⅐RICTOR Correlate
with HMD-PK Activity-ILK, PKC␣, DNA-PK, ATM, and mTOR have all been proposed to play a major role in Akt Ser-473 phosphorylation based on experiments carried out on a variety of different cell types and stimuli (24). We have decided to directly compare the functions of these five proteins in terms of insulin-stimulated Akt Ser-473 phosphorylation in a single cell type, 3T3-L1 adipocytes. Previously, we have shown that LDM and Ext-HiP, purified from 3T3-L1 adipocytes, are greatly enriched in insulin-stimulated HMD-PK activity (27). To determine whether any of these proteins are enriched in LDM and Ext-HiP, we carried out immunoblot analyses on subcellular fractions prepared from serum-starved 3T3-L1 adipocytes ( Fig. 1). DNA-PK was found in the NUC fraction as expected due to its well documented nuclear functions (30). It was, however, more greatly enriched in LDM and Ext-HiP but absent (or below detection) in high density microsomes (HDM), mitochondrial, and CYT fractions. HDM is enriched in endoplasmic reticulum, and LDM contains Golgi markers (42). ATM, another member of the PI 3-kinase-related kinase family, had the same distribution as DNA-PK but was more abundant in NUC than LDM and Ext-HiP as compared with DNA-PK. ILK had a ubiquitous distribution but was slightly more enriched in NUC, LDM, and Ext-HiP fractions. PKC␣ was found almost exclusively in CYT in the basal state. PKCs are cytosolic proteins that translocate to membranes upon hormone stimulation (43). mTOR was greatly enriched in Ext-HiP but was also found in NUC, HDM, and LDM fractions. mTOR has been previously reported to be associated with the endoplasmic reticulum (44) and Golgi (44) and in the nucleus (45). RICTOR, the binding protein of mTOR that is necessary for Ser-473 phosphorylation (31), was very abundant in LDM and less abundant in Ext-HiP but was also present in NUC and HDM fractions.
Next, we wanted to directly correlate HMD-PK activity with the amount of each candidate protein using the cell-free assay. We have previously shown that PIP3-dependent Akt Ser-473 phosphorylation in  In vitro reactions containing 3 mg/ml CYT, 1 mg/ml NUC, 1 mg/ml PM, or 0.37 mg/ml LDM were premixed on ice with 16 nM Akt2 and 10 M PIP3 (sonicated in a mixture of 100 M phosphatidylcholine and 100 M phosphatidylserine). Reactions were initiated with ATP, incubated for 10 min at 37°C, and then subjected to immunoblot analysis using the designated antibodies. our in vitro assay was entirely dependent on exogenous ATP and the time of reaction incubation (27,39). In vitro reactions were performed by combining CYT, NUC, PM, and LDM fractions with purified recombinant Akt2 and ATP in the absence and presence of PIP3. The amount of the particular fraction and the time of incubation were such that the reaction activities were in the linear range (data not shown). Assays were quenched and examined by immunoblot analyses (Fig. 2). The PIP3-stimulated Ser-473 activity in these particular reactions was highest in LDM, followed by NUC and then PM. As we previously observed, no HMD-PK activity was found in CYT (27). The Ser-473 activity correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKC␣. Based on the fact that PKC␣ was almost exclusively localized in CYT, which had no HMD-PK activity, we concluded that PKC␣ was not the HMD-PK in 3T3-L1 adipocytes.
As previously discussed, DNA-PK, ATM, and mTOR are members of the PI 3-kinase-related family of kinases (30). If one of these proteins is indeed HMD-PK in 3T3-L1 adipocytes, then our in vitro Ser-473 phosphorylation activity should be directly sensitive to PI 3-kinase inhibitors. As shown in Fig. 3, PIP3-stimulated Ser-473 activities in both Ext-HiP and LDM were inhibited by wortmannin and LY294002.
Purified DNA-PK Does Not Phosphorylate Full-length Akt2 in a PIP3dependent Manner-Feng et al. (29) has shown that purified DNA-PK (Promega) can phosphorylate a C-terminal Akt peptide containing Ser-473 and two truncated versions of Akt (GST-AKT1-(419 -480), and DPH-Akt2 T309P ) lacking the PH domain. Only the peptide phosphorylation was DNA-dependent. Since it was never shown, we wanted to verify that purified DNA-PK could phosphorylate full-length Akt2 in a PIP3-dependent manner. In vitro reactions, conducted using various combinations of DNA-PK, Akt2, PIP3, and double-stranded DNA, were analyzed by immunoblot analysis (Fig. 4). Control reactions were also performed containing Ext-HiP and LDM. Full-length purified Akt2 was phosphorylated on Ser-473 in a PIP3-dependent manner with either Ext-HiP (Fig. 4A) or LDM (Fig. 4B). The phosphorylation was not stim- , and 10 g/ml salmon sperm DNA were mixed on ice. A, Ext-HiP reactions were initiated with ATP and incubated for 15 min at 37°C. 3ϫ-Ext-HiP refers to reactions in which three times the amount of Ext-HiP (0.6 mg/ml) was used along with Akt2 and PIP3. B, LDM reactions were initiated with ATP and incubated for 5 min at 37°C. 2ϫ-LDM refers to reactions in which twice the amount of LDM (1.5 mg/ml) was used along with Akt2 and PIP3. Samples in A and B were quenched and subjected to immunoblot analysis using Akt Ser-473 phosphospecific or DNA-PK antibodies. C, HMD-PK peptide assays were carried out as described under "Experimental Procedures." Results were quantified as the mean Ϯ S.E. of three independent experiments. The white bars represent results from basal cells, and hatched bars denote results of insulin-stimulated cells. DECEMBER 9, 2005 • VOLUME 280 • NUMBER 49 ulated by DNA. No Ser-473 phosphorylation was observed with Akt2 or Ext-HiP alone. Reactions containing LDM, Akt2, and PIP3 generated two Ser-473 bands of different molecular weights. We have found that the lower band corresponds to endogenous Akt present in LDM, and the upper band is due to the His-tagged recombinant Akt2 (data not shown). In the absence of exogenous Akt2, only the lower Ser-473 band was observed in reactions containing LDM and PIP3. Surprisingly, purified DNA-PK could not phosphorylate full-length Akt2 to any discernible degree in either the presence or absence of PIP3 and DNA. When reactions were analyzed for DNA-PK content, the amount of exogenous DNA-PK greatly exceeded the amount found in Ext-HiP or LDM. To verify that the commercial DNA-PK was active, assays were carried out with the same Ser-473 peptide used by Feng et al. (29) (Fig. 4C). As was reported previously, purified DNA-PK phosphorylated the Ser-473 peptide in a DNA-dependent manner (29). Ext-HiP and LDM could also phosphorylate the peptide; however, the presence of DNA had no statistical effect on the peptide phosphorylation. These results indicate that purified DNA-PK by itself cannot phosphorylate full-length Akt2. It is possible that DNA-PK requires an accessory protein, found in Ext-HiP or LDM, to phosphorylate full-length Akt. The holoenzyme of DNA-PK consists of a 465-kDa catalytic subunit and two Ku antigen subunits (Ku70/Ku80) (30). All three subunits, however, were found in purified DNA-PK, Ext-HiP, and LDM (data not shown). If an accessory protein is required, it is unlikely to be Ku70/80. To determine whether an accessory protein was present in Ext-HiP and LDM and was in excess relative to DNA-PK, we added purified DNA-PK to in vitro reactions containing Ext-HiP/Akt2 (Fig. 4A) or LDM/Akt2 (Fig. 4B) and compared the measured PIP3-stimulated Ser-473 activity with reactions containing Ext-HiP/Akt2 or LDM/Akt2 without exogenous DNA-PK. We found that adding exogenous DNA-PK had no effect. Control reactions containing 3 times the amount of Ext-HiP (3ϫ-Ext-HiP) or twice the amount of LDM (2ϫ-LDM) verified that the reactions were not limited in substrate. Since purified DNA-PK could not phosphorylate full-length Akt2 in a PIP3-dependent manner, either DNA-PK is not HMD-PK or an accessory protein is required but is limiting in LDM or Ext-HiP relative to the amount of DNA-PK.

Phosphorylation of Akt/Protein Kinase B Ser-473 by mTOR⅐RICTOR
HMD-PK Activity Is Not Associated with ILK in LDM-Since Glut 4 vesicles can be easily immunoisolated from LDM of 3T3-L1 adipocytes (46), we attempted to immunoprecipitate DNA-PK, ATM, or ILK from LDM and measure PIP3-stimulated Ser-473 activity in the immunoprecipitates using an in vitro assay. We tested five antibodies for both DNA-PK and ATM and failed to find one that recognized the mouse form of the proteins and that could immunoprecipitate. ILK, however, was successfully immunoisolated from LDM. In vitro assays were then conducted using supernatants and pellets from ILK or control immunoprecipitation reactions. Although we brought down ϳ50% of ILK from LDM, we observed no change in the Ser-473 activity remaining in the supernatant, nor could we measure PIP3-stimulated Ser-473 activity associated with ILK in the pellets (Fig. 5).
siRNA-mediated Depletion of DNA-PK, ATM, or ILK Had No Effect on Insulin-stimulated Akt Ser-473 Phosphorylation in 3T3-L1 Adipocytes-Next we wanted to determine whether suppressing the expression of DNA-PK, ATM, or ILK by siRNA had any effect on insulin-activated Akt Ser-473 phosphorylation in intact 3T3-L1 adipocytes. Using the electroporation method described by Czech and co-workers (6), we could substantially reduce the expressions of DNA-PK (88.7 Ϯ 6.6%), ATM (87 Ϯ 3.5%), and ILK (64 Ϯ 2.6%) (Fig. 6A). Serum-starved cells were then stimulated with insulin and tested for Akt phosphorylation using phosphospecific antibodies. We observed no change in Akt phosphorylation at either Ser-473 or Thr-308 in cells depleted of DNA-PK or ATM compared with cells tested with a negative control siRNA (Fig. 6, B and C). In ILK-depleted cells, we consistently observed a 25% increase in insulin-induced Ser-473 phosphorylation but saw no change in insulin-stimulated Thr-308 phosphorylation (Fig. 6, B and C). The observed increase in Ser-473 phosphorylation was surprising, since we had expected a decrease in insulin-stimulated Ser-473 phosphorylation if ILK was indeed the HMD-PK for Akt. Therefore, based on these results in intact adipocytes and in conjunction with our cell-free experiments, we conclude that DNA-PK, ATM, ILK, and PKC␣ are most likely not the HMD-PK in 3T3-L1 adipocytes.
PIP3-stimulated HMD-PK Activity Was Associated with mTOR⅐RICTOR Complexes from LDM-Next, we focused on mTOR⅐RICTOR by immunoisolating mTOR complexes from LDM and measuring Ser-473 activity with an in vitro assay. PIP3-stimulated Ser-473 activity was found in mTOR complexes immunoprecipitated with antibodies directed against mTOR or RICTOR (Fig. 7A). mTOR, RIC-TOR, and RAPTOR were found in the pellets using the mTOR antibody, whereas the RICTOR antibody brought down mTOR and RICTOR but not RAPTOR. We found no commercial RAPTOR antibody that could successfully immunoprecipitate RAPTOR. Nevertheless, our results indicate that complexes containing mTOR and RICTOR are capable of phosphorylating Akt on Ser-473 in a PIP3-dependent manner. The in vitro reactions were also probed for the presence of DNA-PK, ATM, and ILK. We found no detectable amounts of these three proteins in the mTOR or RICTOR immunoprecipitation pellets, although all three were present in total LDM (Fig. 7B).
To help rule out the possibility that Akt was phosphorylated on Ser-473 by a kinase other than mTOR that happens to co-immunoprecipitate in our system, we immunoprecipitated mTOR from LDM and then measured HMD-PK activity in the presence of PI 3-kinase inhibitors and staurosporine (Fig. 8). PIP3-stimulated Ser-473 activity was inhibited with wortmannin and LY294002 but not staurosporine. Our results are consistent with those of Sabatini and co-workers for mTOR (31).
siRNA-mediated Depletion of RICTOR Inhibited Insulin-stimulated Akt Ser-473 Phosphorylation in 3T3-L1 Adipocytes-Unfortunately, we were unsuccessful at silencing mTOR expression in 3T3-L1 adipocytes using six different commercially available siRNAs. Regardless, it has been reported that almost complete knockdown of mTOR is required to observe any effect on insulin-stimulated Akt Ser-473 phosphorylation (31), a result most likely due to the feedback inhibition of insulin signaling caused by serine phosphorylation of IRS-1 by mTOR/RAPTOR (36). To circumvent this problem, we attempted to suppress RICTOR expression in order to affect insulin-stimulated Akt Ser-473 phosphorylation. We were able to substantially silence by electroporated siRNA (85% Ϯ 3.5%) RICTOR expression with no effect on mTOR levels (Fig.  9A). Serum-starved adipocytes were stimulated with insulin and then analyzed for Akt phosphorylation using phosphospecific antibodies (Fig. 9, B and C). Insulin-activated Akt phosphorylation of Ser-473 and Thr-308 were suppressed 87 Ϯ 3.2 and 55 Ϯ 9.35%, respectively, when RICTOR expression was reduced with siRNA. These results were also confirmed using a pool of two independent RICTOR siRNAs (target regions 470 -490 and 903-923; Qiagen) whose sequences were differ-ent from the ones used above (results not shown). The tight correlation between RICTOR expression levels and the amount of insulin-stimulated Akt Ser-473 phosphorylation was further demonstrated by a RIC-TOR siRNA dose response experiment (Fig. 9D). Based on our cell-free and siRNA results, we conclude mTOR complexed with RICTOR is the HMD-PK in 3T3-L1 adipocytes.

DISCUSSION
DNA-PK (29), ATM, (47) ILK (48,49), PKC␣ (50), and mTOR (31) have all been recently identified as candidates for the HMD-PK for Akt. These studies, however, were all conducted in different cell types using a variety of stimuli. Our report is the first to directly address all five candidates in a single cell type, 3T3-L1 adipocytes, activated using a single stimulus, insulin. We selected 3T3-L1 adipocytes because these FIGURE 6. siRNA-mediated depletion of DNA-PK, ATM, or ILK has no effect on insulin-stimulated Akt Ser-473 phosphorylation in 3T3-L1 adipocytes. Control, DNA-PK, ATM, and ILK siRNAs were electroporated in differentiated 3T3-L1 adipocytes (day 5) as described under "Experimental Procedures." 72 h after electroporation, cells were serum-starved for 2 h, activated or not with 100 nM insulin for 1.5 min, and then harvested in Buffer A containing 1% SDS. Equal amounts of lysates were subjected to immunoblot analysis using the designated primary antibodies. A, immunoblot analysis using DNA-PK-, ATM-, and ILK-specific antibodies. B, immunoblot analysis using Akt Ser-473 and Akt-Thr-308 phosphospecific antibodies. C, quantification of the mean Ϯ S.E. of three independent experiments. White bars represent results from basal cells, and hatched bars denote results of insulin-stimulated cells. Phosphorylations were normalized to ϩinsulin controls. *, phosphorylations differed significantly (p Ͻ 0.05) compared with insulin-stimulated control samples. An unpaired Student's t test was used in all statistical analyses. DECEMBER 9, 2005 • VOLUME 280 • NUMBER 49 cells are very responsive in terms of insulin-stimulated glucose transport (51), a process in which Akt activation is a critical step (3)(4)(5)8).

Phosphorylation of Akt/Protein Kinase B Ser-473 by mTOR⅐RICTOR
The original identification of DNA-PK as the Ser-473 kinase was very surprising, since DNA-PK was already well documented to possess several DNA-related functions that take place in the nucleus (30). To carry out these functions, double-stranded DNA is required to co-localize substrates with DNA-PK through their interactions with Ku proteins and to activate DNA-PK. It is possible that our apparent enrichment of DNA-PK in LDM and Ext-HiP is entirely due to the disruption of nuclei that may occur during the homogenization process (Fig. 1). What argues against this is that Feng et al. (29) have shown by confocal microscopy that a significant fraction of DNA-PK is found in the cytosol and at the membrane in addition to the nucleus in 3T3-L1 fibroblasts and M059K cells. The strongest data that supported the idea that DNA-PK can function as a Ser-473 kinase were siRNA knockdown experiments in HEK293 cells, the lack of significant insulin-induced Ser-473 phosphorylation in DNA-PK-deficient M059J glioblastoma cells, and rescue experiments with the M059J/Fus1 cells (29). The residual amount of insulin-activated Ser-473 phosphorylation in M059J cells has been recently shown to be due to mTOR⅐RICTOR (31). Nevertheless, mTOR⅐RICTOR cannot explain why Ser-473 phosphorylation is so low in the absence of DNA-PK in M059J cells. It is possible that in certain cell types under certain conditions, DNA-PK does function as the primary HMD-PK for Akt. Recently, DNA-PK has been implicated in the activation of Akt by CpG-DNA (52). Stimulation of Ser-473 phosphorylation by CpG-DNA is markedly reduced in bone marrow-derived macrophages from mice lacking DNA-PK. Our results, however, indicated that DNA-PK could not phosphorylate full-length Akt2, although FIGURE 7. PIP3-stimulated HMD-PK activity associates with mTOR⅐RICTOR complexes from LDM. 500 g of LDM were immunoprecipitated (IP) with control (CON)-, mTOR-, or RICTOR-specific polyclonal antibodies as described under "Experimental Procedures." In vitro reactions containing pellets from the immunoprecipitates, Akt2, and with or without 10 M PIP3 (sonicated in a mixture of 100 M phosphatidylcholine and 100 M phosphatidylserine) were mixed with ATP and incubated for 10 min at 37°C. A, reactions were quenched and subjected to immunoblot analysis using the designated antibodies. B, reactions from A were also subjected to immunoblot analysis using DNA-PK-, ATM-, and ILK-specific antibodies. Total LDM provides a positive control for the DNA-PK, ATM, and ILK antibodies. it could phosphorylate truncated versions of Akt (29) (Fig. 4). Therefore, if DNA-PK can really phosphorylate Ser-473 in cells, it most likely would require an accessory factor. The necessity of an accessory factor for DNA-PK to phosphorylate Akt would not be unexpected, since its related family member mTOR must form a complex with either RAP-TOR or RICTOR to phosphorylate p70S6 kinase (35) or Akt (31), respectively. In the case of DNA-PK, this accessory factor would be necessary to shift its phosphorylation preference from (S/T)Q motifs (53) to the FXXF(S/T)(Y/F) found in Akt (20). In fact, it was recently shown that purified DNA-PK could phosphorylate Akt2 in the presence FIGURE 9. siRNA-mediated depletion of RICTOR inhibits insulin-stimulated Akt Ser-473 phosphorylation in 3T3-L1 adipocytes. Control (20 nmol) and RICTOR (50 nmol) siRNAs were electroporated in differentiated 3T3-L1 adipocytes (day 5) as described under "Experimental Procedures." 72 h after electroporation, cells were serum-starved for 2 h, activated or not with 100 nM insulin for 1.5 min, and then harvested in Buffer A containing 1% SDS. Equal amounts of lysates were subjected to immunoblot analysis using the designated primary antibodies. A, immunoblot analysis using RICTOR-, mTOR-, and Akt-specific antibodies. B, immunoblot analysis using Akt Ser-473 and Akt-Thr-308 phosphospecific antibodies. C, results in panel B were quantified as the mean Ϯ S.E. of three independent experiments. White bars represent results from basal cells, and hatched bars denote results of insulin-stimulated cells. Phosphorylations were normalized to ϩinsulin controls. *, phosphorylations differed significantly (p Ͻ 0.05) compared with insulin-stimulated control samples. An unpaired Student's t test was used in all statistical analyses. D, RICTOR siRNA dose response upon insulin-stimulated Akt Ser-473 phosphorylation. The amounts (in nmoles) of control and RICTOR siRNAs are indicated in parentheses. Immunoblot analyses using RICTOR antibodies and Akt Ser-473 phosphospecific antibodies were quantified as the mean Ϯ S.E. of three independent experiments. of a synthetic CpG oligonucleotide (CpG-OGN) but not in its absence (52). It is clear, however, from the lack of an effect on insulin-stimulated Akt Ser-473 phosphorylation when DNA-PK was knocked down by almost 90% with siRNA in 3T3-L1 adipocytes, that DNA-PK does not significantly contribute to HMD-PK activity in fat cells in response to insulin (Fig. 6). Our results, however, do not address the function of DNA-PK in other cell types.
ATM, like DNA-PK, is a kinase involved in DNA repair (54) that has also been implicated in Akt Ser-473 phosphorylation (47). It has been reported that overexpression of ATM robustly increased Ser-473 phosphorylation, whereas knockdown by siRNA significantly decreased it. Thr-308 phosphorylation was not affected in either case. ATM, however, cannot directly phosphorylate Akt on Ser-473 in vitro, suggesting that it may play an indirect role in Ser-473 phosphorylation. Our results in 3T3-L1 adipocytes indicated that ATM most likely has little if any role in Ser-473 phosphorylation, direct or indirect. ATM was not very abundant in LDM or Ext-HiP, the two fractions that possess the highest Ser-473 activity ( Figs. 1 and 2). In addition, a greater than 85% reduction in the expression of ATM by siRNA had no effect on the phosphorylation of Ser-473 in response to insulin (Fig. 6).
Dedhar and co-workers (48,49,55) have published multiple reports providing evidence that ILK can function as the Akt Ser-473 kinase. Recombinant ILK can phosphorylate Akt at Ser-473 in vitro (55). A kinase-dead ILK has been shown to act as a dominant negative suppressing endogenous Ser-473 phosphorylation activity (49). Knockdown experiments by siRNA and knockouts using the Cre-LoxP system markedly reduced Ser-473 phosphorylation (48). Despite these data, there are other studies that indicate ILK may not be the physiological Akt Ser-473 kinase. It was reported that in ILK-deficient fibroblasts (56) and chondrocytes (57), Ser-473 phosphorylation was normal. Other groups have not been able to phosphorylate Akt at the HM site in vitro (28,58,59). There is even uncertainty that ILK is a functional kinase, since several critical residues normally found in the catalytic domain of protein kinases are not conserved (59). Thus, what role ILK has in terms of the phosphorylation of Akt on Ser-473 may be indirect. In 3T3-L1 adipocytes, our data indicated that ILK was not the HMD-PK for Akt. We saw no change in the level of PIP3-stimulated Akt Ser-473 phospho-rylation activity in LDM after removing more than half the ILK and saw no corresponding appearance of activity in the pellets after immunoprecipitation (Fig. 5). Similarly, after reducing the expression of ILK by ϳ65% with siRNA, we actually observed a 25% increase rather than a decrease in insulin-activated Akt Ser-473 phosphorylation (Fig. 6). Further study is necessary to determine whether the 25% increase in Ser-473 phosphorylation is of physiological significance.
PKC␣ is activated by diacylglycerol and calcium (43) and has been implicated in Ser-473 phosphorylation (50). This PKC isoform can phosphorylate Akt on Ser-473 in vitro in the presence of lipid and calcium as well as undergo IGF-1-stimulated translocation to membranes in endothelial cells. Overexpression of PKC␣ increased Ser-473 phosphorylation, whereas dominant-negative PKC␣ constructs or reducing the expression by siRNA decreased Ser-473 phosphorylation. We found that in 3T3-L1 adipocytes, PKC␣ was almost entirely cytosolic and absent from compartments with Ser-473 phosphorylation activity (Figs. 1 and 2). In addition, our cell-free assays were all conducted in the absence of calcium, since 1 mM EGTA was included in the reaction buffer. Our conclusion that PKC␣ is not involved in the Ser-473 phosphorylation of Akt in adipocytes is in agreement with an earlier report that Akt phosphorylation at Ser-473 actually increased when PKC␣ was knocked out in fat and muscle (60). The authors of the knock-out study concluded that PKC␣ inhibits insulin signaling by phosphorylating IRS proteins on serine residues resulting in the suppression of insulin-stimulated tyrosine phosphorylation of IRS proteins by the insulin receptor. Interesting with regard to PKC␣, Sarbassov et al. (38) reported that mTOR⅐RICTOR complex can regulate the organization of the actin cytoskeleton and they have proposed that this regulation is mediated via the phosphorylation of PKC␣. PKC␣ does not, however, form a stable complex with mTOR⅐RICTOR in detergent-solubilized cells (31). In addition, PKC␣ immunoprecipitates prepared from detergent-solubilized cells did not phosphorylate Akt Ser-473 in an in vitro kinase assay.
Based on our results, we have concluded that mTOR⅐RICTOR is the HMD-PK for Akt in 3T3-L1 adipocytes. mTOR⅐RICTOR was greatly enriched in fractions that contain the highest Ser-473 activity (Figs. 1  and 2). mTOR⅐RICTOR complexes, purified by immunoprecipitation of LDM with mTOR or RICTOR antibodies, had PIP3-stimulated Ser-473

Phosphorylation of Akt/Protein Kinase B Ser-473 by mTOR⅐RICTOR
phosphorylation activity (Fig. 7A) and did not contain DNA-PK, ATM, or ILK (Fig. 7B). In addition, the Ser-473 activity associated with mTOR⅐RICTOR complexes was sensitive to PI 3-kinase inhibitors but not staurosporine (Fig. 8), the same drug sensitivity has been shown for insulin-stimulated Akt Ser-473 phosphorylation (16,61). Decreasing the expression of RICTOR by siRNA substantially inhibited insulinstimulated Akt Ser-473 and to a lesser extent Thr-308 phosphorylation in intact adipocytes (Fig. 9). Effects on both Ser-473 and Thr-308 phosphorylations are in agreement with the model that the phosphorylation of Ser-473 provides a docking site to recruit phosphoinositide-dependent kinase 1 in order to phosphorylate Thr-308 (20). It should also be pointed out that the basal activity we observed in the Ser-473 peptide assays conducted with Ext-HiP or LDM was most likely due to mTOR⅐RICTOR complexes in these fractions and not due to DNA-PK (Fig. 4C). All of our results are entirely consistent with those published by Sabatini and co-workers (31). It has also been recently reported that treating 3T3-L1 adipocytes with rapamycin actually increases insulinstimulated Akt Ser-473 phosphorylation (36). This result can be fully explained in terms of feedback inhibition of insulin signaling caused by serine phosphorylation of IRS-1. Rapamycin inhibited insulin activation of p70S6 kinase, decreased Ser 636/639 IRS-1 phosphorylation, increased PIP3 levels, and resulted in an increase in Akt Ser-473 phosphorylation (36).
The model proposed by Sabatini and co-workers (31) also explains how mTOR can be found both upstream and downstream of Akt in the insulin-stimulated PI 3-kinase pathway (Fig. 10). mTOR, when complexed to RICTOR and G␤L acts as HMD-PK activating Akt through the phosphorylation of Ser-473. Akt also activates mTOR/RAPTOR, either directly or through the phosphorylation of the tumor suppressor, TSC2, leading to the stimulation of the p70S6 kinase/4EBP1 pathway (62).
Interestingly, Sabatini and co-workers (31) have also reported that the Ser-473 kinase activity of mTOR⅐RICTOR was markedly stimulated by serum in vivo. Kinase assays conducted on immunoprecipitates isolated from 0.3% CHAPS-solubilized serum-treated HeLa cells were significantly higher than those isolated from basal cells. Activation of HMD-PK by serum would disagree with the assumption by Hemminngs that the Ser-473 kinase is constitutively active at the plasma membrane (15,63,64). It is thought that binding of Akt to PIP3, formed in PM in response to insulin, has two effects, it co-localizes Akt with a constitutively active HMD-PK (15,63) and also induces a conformational change in Akt that allows for Ser-473 and Thr-308 phosphorylation (65) (Fig. 10). Therefore, insulin enhances Ser-473 phosphorylation through effects on Akt itself as opposed to effects on HMD-PK. We, in fact, have observed no difference in HMD-PK activity in cell-free assays conducted using Ext-HiP (or LDM) purified from basal and insulin-treated adipocytes, 3 which tend to support the idea that insulin does not activate HMD-PK directly. One explanation for the apparent discrepancy is that the observed serum-induced increase in HMD-PK activity reported by Sabatini and co-workers (31) is actually due to the presence of PIP3 in the immunoprecipitates when mTOR complexes were isolated from serum-treated cells. 0.3% CHAPS is not a high enough detergent concentration to completely solubilize membranes. In fact, these same authors have reported that the mTOR⅐RICTOR complex was not stable under conditions (1% Triton X-100) typically used to solubilize membranes.
In summary, our results indicate that mTOR⅐RICTOR, is responsible for insulin-induced Akt Ser-473 phosphorylation in 3T3-L1 adipocytes.
Our results do not address whether DNA-PK, ATM, ILK, or PKC␣ can function as HMD-PK for Akt in other cell types under certain conditions.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Amino Acid-dependent Transfer RNA Affinity in a Class I Aminoacyl-tRNA Synthetase*

Steady-state and transient kinetic analyses of glutaminyl-tRNA synthetase (GlnRS) reveal that the enzyme discriminates against noncognate glutamate at multiple steps during the overall aminoacylation reaction. A major portion of the selectivity arises in the amino acid activation portion of the reaction, whereas the discrimination in the overall two-step reaction arises from very weak binding of noncognate glutamate. Further transient kinetics experiments showed that tRNAGln binds to GlnRS ∼60-fold weaker when noncognate glutamate is present and that glutamate reduces the association rate of tRNA with the enzyme by 100-fold. These findings demonstrate that amino acid and tRNA binding are interdependent and reveal an important additional source of specificity in the aminoacylation reaction. Crystal structures of the GlnRS·tRNA complex bound to either amino acid have previously shown that glutamine and glutamate bind in distinct positions in the active site, providing a structural basis for the amino acid-dependent modulation of tRNA affinity. Together with other crystallographic data showing that ligand binding is essential to assembly of the GlnRS active site, these findings suggest a model for specificity generation in which required induced-fit rearrangements are significantly modulated by the identities of the bound substrates.

The high fidelity of protein synthesis in living cells arises as a consequence of specificity at three distinct steps in the pathway: aminoacyl-tRNA formation, selection of aminoacyl-tRNA by elongation factor Tu, and ribosomal proofreading of the codon-anticodon interaction (1)(2)(3). Accuracy at the first aminoacyl-tRNA synthesis step is accomplished by the aminoacyl-tRNA synthetases, which attach amino acids to the corresponding tRNA species in a two-step reaction. Amino acids are first activated to their corresponding adenylates by harnessing the energy of ATP hydrolysis, generating pyrophosphate. In the second step, the oxygen nucleophile at the 2Ј-or 3Ј-ribose position of the 3Ј-terminal A76 tRNA nucleotide attacks the carbonyl carbon of the mixed anhydride intermediate, forming aminoacyl-tRNA with release of AMP (1). Formation of only cognate aminoacyl-tRNA species re-quires that the enzymes be specific for both amino acid and tRNA substrates.
Although all tRNAs possess largely similar L-shaped tertiary structures, significant capacity for discrimination among these species arises from direct recognition of base-specific functional groups at key nucleotide positions (identity determinants), as well as by an indirect readout process in which selective enzyme interactions are made with groups in the sugar-phosphate backbone or nonspecific portions of the bases (4). The existence of 20 parallel aminoacyl-tRNA systems in vivo promotes further discrimination by competition (5). In contrast, amino acid specificity relies on a much smaller number of possible interactions between the enzyme and substrate; moreover, many of the amino acids possess similar structural and chemical properties. In some difficult cases (e.g. isoleucine versus valine, or valine versus threonine), tRNA synthetases cannot sufficiently discriminate among certain amino acids during the two-step synthesis reaction, and rely instead on an additional editing mechanism by which misacylated tRNA is hydrolyzed in a spatially separate editing active site (1).
Glutaminyl-tRNA synthetase (GlnRS) 1 is a class I tRNA synthetase that achieves amino acid selectivity without use of an editing mechanism. Escherichia coli GlnRS synthesizes Gln⅐tRNA Gln 10 7 -fold more efficiently than Glu⅐tRNA Gln , corresponding to discrimination of 10.1 kcal/mol in favor of glutamine versus the nearly isosteric glutamate (6). Crystal structures of GlnRS have been solved in a ternary complex bound to tRNA and ATP, as well as in quaternary complexes bound to either cognate glutamine or noncognate glutamate in the presence of an ATP analog. The quaternary structures showed that the two amino acids bind in distinct orientations in the active site cleft (6,7) and that a key arginine residue (Arg-30) may function as a negative determinant to drive glutamate binding into a nonproductive orientation via an apparently strong ionic interaction. This crystallographic finding predicts that a significant component of the strong amino acid selectivity might arise at the chemical rather than the binding step of the reaction.
Evidence exists suggesting that amino acid and tRNA binding to GlnRS are modulated by induced fit: mutually induced conformational changes in enzyme and substrates occur during initial binding and en route to a productive quaternary groundstate complex. GlnRS is one of four class I tRNA synthetases that require the presence of tRNA for catalysis of adenylate formation, suggesting that tRNA is required for proper active site assembly (8). Supporting this notion, comparisons of the unliganded and tRNA-bound GlnRS structures shows that portions of both the amino acid and ATP binding sites are misoriented with respect to each other in the absence of tRNA (9,10). Furthermore, fluorescence spectroscopy reveals ATP-dependent differences in binding of tRNA Glu and tRNA Gln to GlnRS that are suggestive of an induced-fit process (11,12). Finally, kinetic data on modified GlnRS⅐tRNA complexes show that tRNA mutants in the distal anticodon and tertiary core regions can cause large decreases in both the k cat for aminoacylation and the K m for glutamine (13,14). Pre-steady-state kinetics further demonstrated that aminoacylation of the tRNA Gln anticodon mutant U35A is decreased by 30-fold at the chemical step of the reaction and that glutamine binding affinity in the U35A mutant complex is weakened by 20-fold (15). These data demonstrate definitively that anticodon binding signals are transmitted some 40 Å to the GlnRS active site.
To explore the detailed origins of amino acid specificity in GlnRS, we performed steady-state and pre-steady-state kinetic studies utilizing both the conventional ATP-PP i exchange reaction, as well as a newer assay that relies upon internal 32 P labeling of tRNA (16). This assay is highly sensitive and permits use of very high amino acid concentrations, a requirement for rigorous measurements of noncognate reactions in which ligand affinities are weak. We find that the specificity of GlnRS against glutamate in the two-step reaction is partitioned between the binding and catalytic steps of the reaction, with a greater component at the binding step. Furthermore, the presteady-state kinetic analysis uncovers a significant coupling between the amino acid and tRNA affinities of the enzyme, in which the identity of the bound amino acid (cognate or noncognate) controls how tightly tRNA Gln binds. This finding suggests that induced-fit structural pathways connecting the amino acid and tRNA binding sites may contribute to enzyme specificity. Thus, features of the enzyme architecture that control the required ligand-induced conformational changes may have a further role in helping to reject noncognate substrates. To date, GlnRS is the only tRNA synthetase for which noncognate aminoacylation has been examined by both crystallographic and pre-steady-state kinetic analyses. Nonetheless, the common presence of induced-fit rearrangements among many tRNA synthetases, as revealed by x-ray structures in the apo-and liganded states, suggests that this mechanism may be quite general to the enzyme family.

EXPERIMENTAL PROCEDURES
Enzyme Purifications-T7 RNA polymerase was purified as described from E. coli BL21 cells containing the plasmid pAR1219 (6,17). Purified enzyme was stored at Ϫ20°C in a buffer containing 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 50% glycerol, 20 mM sodium phosphate (pH 7.8). A His 6 -C-terminal-tagged GlnRS was constructed by inserting the glnS gene after the T7 promoter of the expression vector pSJW1 (18). The His-tagged GlnRS was expressed in BL21-DE3(pLysS) by induction with 1 mM isopropyl 1-thio-␤-D-galactopyranoside at A 600 between 0.4 and 0.6. Cells were resuspended in a buffer containing 0.5 M NaCl, 50 mM HEPES (pH 7.2), 10 mM imidazole and were disrupted by French press. 100 mM phenylmethylsulfonyl fluoride, 100 mM benzamidine, 50 l of RQ1 DNase (Promega), and 15 mM MgCl 2 were added to the lysate, and the mixture was stirred at room temperature for 20 min. Lysed cells were then centrifuged for 30 -45 min at 15,000 rpm, and the pellet was discarded. Enzyme was purified on a 5-ml nickel column (Amersham Biosciences) equilibrated in 10 mM imidazole, 20 mM HEPES (pH 7.2), 1 mM ␤-mercaptoethanol, and 0.5 M NaCl; the elution buffer was identical to the equilibration buffer except for the inclusion of 120 mM imidazole. Eluted protein was dialyzed into storage buffer containing 50% glycerol, 20 mM potassium phosphate (7.4), 50 mM KCl, 2 mM DTT for 6 -12 h. His-tagged GlnRS was recovered at better than 99% purity as judged by SDS-PAGE and was stored at high concentration at Ϫ20°C, as described for the native enzyme (6). GlnRS concentration was determined by absorbance (⌭ 280 (1 mg/ml) ϭ 1.06). Kinetic parameters of the His-tagged GlnRS were indistinguishable from that of native GlnRS within experimental error.
tRNA Synthesis and Purification-E. coli G-1 tRNA 2 Gln (containing a catalytically neutral U1G mutation to promote efficient transcription initiation) was transcribed in high yield from a synthetic DNA template. The template was constructed from complementary synthetic oligonucleotides containing a short central overlapping region, which were extended to form the full-length duplex by treatment with the Klenow fragment of E. coli DNA polymerase I (19). The DNA template incorporated 2Ј-O-methyl sugars at the two 5Ј-nucleotides in the noncoding DNA strand, resulting in a high proportion of enzymatically active tRNA transcripts. The reactions were then loaded onto a 5-ml DE-52 (Whatman) column, eluted, precipitated with ethanol, dried, and stored at Ϫ20°C. Prior to use the tRNA was resuspended in and dialyzed against highly purified water.
ATP-PP i Exchange Assay-The reaction mixture for the ATP-PP i exchange assay consisted of 100 mM Mes-KOH (pH 6.0), 5 mM ATP, 20 mM MgCl 2 , 25 mM KCl, 4 mM DTT, 1 mM [ 32 P]PP i (10 -50 Ci/ml). K m and k cat for glutamine and glutamate were determined by varying concentrations of these substrates from 0.05-50 mM and from 0.05-1.2 M, respectively. The concentrations of GlnRS used were 5 nM for glutamine activation reactions, and 100 nM for glutamate activation reactions. In both cases, the concentration of tRNA Gln was 10 M. The reactions were stopped by adding 1.5 l of the reaction mixture to 3.0 l of a solution containing 400 mM sodium acetate (pH 5.0), 0.1% SDS. 2.0 l of this mixture was spotted on polyethyleneimine cellulose plates that had been prewashed in water. Thin layer chromatography, using 750 mM KH 2 PO 4 (pH 3.5) and 4 M urea as developing buffer, was employed for separation of [ 32 P]ATP from [ 32 P]PP i and [ 32 P]P i as previously described (20). The dried chromatogram was exposed overnight to an image plate and was quantified on a Storm 840 PhosphorImager (Amersham Biosciences). Kinetic parameters were obtained from at least three independent measurements. Initial velocities obtained by time-course analyses were plotted against substrate concentration and fit to the Michaelis-Menten equation with KaleidaGraph. K m and k cat were determined directly from these plots.
Aminoacylation Assay and Steady-state Reactions-Aminoacylation assays were carried out by a highly sensitive assay that has been recently described (6,15,16). Full-length tRNA Gln substrate prepared by in vitro transcription was 32 P-labeled at the 3Ј-terminal internucleotide linkage using ␣-32 P-labeled ATP and the exchange reaction of tRNA nucleotidyltransferase (6). To prepare the tRNA sample for use in kinetic assays, labeled and unlabeled substrate were mixed to the appropriate final concentration, heated at 65°C for 3 min, and then cooled at room temperature in the presence of 7.5 mM MgCl 2 . All reactions were quenched in a buffer containing 0.1% SDS, 0.15 M sodium acetate (pH 5.2). P1 nuclease digestions were then performed by adding 1-5 l of the reaction mixture to a microtiter well containing 3-5 l of 0.1 mg/ml P1 nuclease (Fluka), 0.15 M sodium acetate (pH 5.2) and incubating 10 min at ambient temperature. Aminoacylated tRNA (as 3Ј-aminoacylated A76) and nonreacted substrate (as unmodified AMP) were separated by TLC and quantitated by phosphorimaging analysis, as described before (6).
Plateau aminoacylation levels were determined directly by the ratio of aminoacyl-A76 and unmodified A76 after P1 nuclease treatment and TLC separation (6). All steady-state kinetic reactions were performed at 37°C in a buffer containing 10 mM ATP, 5 mM DTT, 50 mM Tris (pH 7.3), and 15 mM MgCl 2 . Concentration ranges of glutamine and tRNA Gln used to determine Michaelis parameters for cognate glutaminylation reactions were as reported (15). To determine k cat and K m [tRNA Gln ] for glutamylation reactions, the reactions were performed at 0.3 M GlnRS and 2 M glutamate. The tRNA Gln concentration was varied between 1.4 and 56 M. For both cognate and noncognate steady-state reactions, time courses for product formation were replotted using Eadie-Hofstee and hyperbolic (Equation 4) analysis to derive k cat and K m .
Rapid Chemical-quench Kinetics-Single-turnover reactions were performed under conditions of 5-fold enzyme excess under solution and temperature conditions identical to the steady-state reactions. Measurements of cognate glutaminylation reactions were performed using a rapid chemical-quench flow apparatus (Kintek RQF-3) as described elsewhere (15). Noncognate misaminoacylation reactions were slow enough to allow sampling by hand. All reactions were quenched in 50 l of 400 mM sodium acetate (pH 5.2), 0.1% SDS. 8 -10 time points were collected for each k chem determination. The amino acid K d values under single-turnover conditions were determined by titrating the concentration of the amino acid while maintaining saturating levels of ATP and tRNA. The glutamine K d value in cognate reactions was previously determined as described (15). For noncognate glutamylation, the K d for glutamate was evaluated at saturating concentrations of the other substrates: 10 mM ATP, 30 M tRNA Gln , and 150 M GlnRS. Titrations to assess K d for tRNA Gln were performed at 10 mM ATP and either 10 mM glutamine or 2 M glutamate. All data were acquired in triplicate. Burst kinetics analyses in pre-steady-state experiments for noncognate glutamylation were performed similarly to those for cognate reactions (15), at concentrations of 0.3 M GlnRS, 27 M tRNA Gln , 2 M glutamate, and 10 mM ATP.
Pulse-chase Experiments-Pulse-chase trap experiments for cognate glutaminylation were performed as described above for the singleturnover rapid quench experiments, with the following exceptions. The concentration of tRNA Gln was kept constant at 13 nM, whereas the concentration of GlnRS was varied between 40 and 120 nM. Bulk E. coli tRNA, purified from overexpression strain JM109 containing plasmid pRS3 (21), was dissolved in 10 mM Pipes (pH 7.2), 10 mM MgCl 2 and used as the quench solution. The final tRNA concentration in quenched reactions was 200 M. Directly after adding the bulk tRNA, 15-45 s elapsed before 5 l of the reaction mixture was aliquoted into 5 l of a solution of P1 nuclease prepared as described above. Noncognate glutamylation pulse-chase experiments were performed as described above for the single-turnover glutamylation reactions, except that the tRNA Gln concentration was kept at either 60 or 130 nM, whereas the concentration of GlnRS was varied between 0.16 and 13 M.
Data Analysis-Data were fit with KaleidaGraph (Synergy Software) and/or Scientist (MicroMath Inc.) curve-fitting programs using the equations below (22,23). Standard errors reported are the standard deviations of all data used in each fit. Pre-steady-state time courses for glutaminylation and glutamylation reactions were fit to either a single first-order exponential (Equation 1) or two consecutive first order reac- where O is the amplitude, t is time, and AA is the fraction of tRNA Gln aminoacylated, or % aminoacylation as described above.
The rate constants k on , k off , and k chem are to be interpreted according to the reaction depicted in the simplified reaction for data analysis shown in Fig. 3, where k on and k off refer to the association and dissociation rates, respectively, for either ATP, amino acid, or tRNA Gln . k chem is a composite intrinsic rate constant for the two-step aminoacylation reaction.
The amplitude of aminoacylation was determined by the fit for each time course. Burst kinetics values were fit by Equation 3.
The kinetic K d for tRNA Gln in the presence of glutamate was calculated by replotting GlnRS concentration against the k chem for that concentration. These data were then fit to either a hyperbolic (Equation 4) or quadratic binding equation (Equation 5) according to the concentration of tRNA Gln used in the reactions: Here E is the concentration of GlnRS, T is the concentration of tRNA Gln , and O is the amplitude. Time courses for pulse-chase experiments, performed under conditions where enzyme is in molar excess to tRNA, were fit to a singleexponential function. The enzyme concentration was then replotted against the observed rate (k obs ) for catalysis (Equation 6). Under circumstances where k off is very small, the slope k obs /[E] reduces to the rate of association (k on ). However, if the magnitudes of k off and k chem are similar, then the value of k obs /[E] sets a lower limit on the rate of association.
Kinetic data exhibiting a prominent lag phase was fit using the reaction scheme described above, using the program Scientist. In these reactions ATP and glutamine saturate the enzyme complex, and the tRNA Gln binding equilibria account for the rate-limiting interactions that are indicated by appearance and disappearance of the lag. Global fits using nonlinear regression analysis were used to define values for the amplitude and for each of the distinct rate constants k on , k off , and k chem reported in Table II.

Discrimination against Glutamate at the Amino Acid Activation
Step-To evaluate the contribution of the aminoacyl adenylate synthesis step to the overall accuracy of glutaminylation, the rate of exchange of 32 P-labeled pyrophosphate into ATP was measured in the presence of either cognate glutamine or noncognate glutamate (Table I). Unlike most tRNA synthetases, GlnRS does not synthesize the aminoacyl adenylate intermediate in the absence of tRNA. We therefore first searched for reaction conditions in which transfer of amino acid to the tRNA would be sufficiently slow to permit measurement of ATP-PP i exchange. This revealed that the proportion of radiolabeled ATP synthesized increases substantially at pH 6.0, where aminoacylation efficiency is reduced (Fig. 1). This is the case even though the velocity of ATP-PP i exchange also decreases at the lower pH values, as may be seen from the fact that reactions at lower pH take a longer time to reach plateau levels. Reactions at pH 6.0 were conducted in the presence of either glutamine or noncognate glutamate, and the steadystate velocity of ATP formation was monitored ( Fig. 1 and Table I). Interestingly, the K m of 3.2 mM for glutamine in this reaction is 16-fold higher than the K m of 0.2 mM measured for the two-step glutaminylation reaction (15). The elevation in K m may indicate a change in the glutamine binding affinity at lower pH; alternatively, because the reaction mechanisms differ in the two assays, the distinction may reflect instead an alteration in other kinetic parameters (22). In the presence of tRNA Gln , rapid exchange between ATP and PP i occurred with k cat of 47 s Ϫ1 (Table I), significantly faster than the steadystate rate of Gln⅐tRNA Gln formation in the two-step reaction (k cat ϭ 3.2 s Ϫ1 (14)). This is expected given that dissociation of Gln⅐tRNA Gln is known to be the rate-limiting step in the aminoacylation pathway (15).
The ATP-PP i exchange reaction was also performed in the presence of glutamate. Significantly impaired synthesis of glutamyl adenylate was observed in comparison to the cognate reaction (Table I). The K m for glutamate is increased by 10 2fold compared with glutamine, whereas k cat is decreased by at most 10 3 -fold. It is known that the level of discrimination against glutamate in the overall aminoacylation reaction is 10 7 -fold, as assessed by the relative values of k cat /K m (6). Thus, a major component (Ͼ4 ϫ 10 4 -fold) of this selectivity arises in the first activation step of the reaction.
Partitioning of Amino Acid Discrimination between Binding and Catalytic Steps-A novel, highly sensitive assay for tRNA aminoacylation was applied to evaluate the kinetic origins of amino acid discrimination by E. coli GlnRS. The assay is based on 32 P-labeling at the 3Ј-internucleotide linkage of the tRNA (16). After aminoacylation, the labeled tRNA is degraded by P1 nuclease, resulting in a mixture of labeled aminoacyl-AMP (product) and labeled AMP (unreacted substrate). These two compounds are then separated by TLC and quantified by image  analysis (Fig. 2). Previously, we used the new assay to determine k cat /K m for cognate glutamination and for formation of misacylated Glu⅐tRNA Gln by GlnRS (6). These data showed that GlnRS discriminates against noncognate glutamate by 10 7 -fold, or 10.1 kcal/mol in the transition state for aminoacylation. Steady-state parameters for glutaminylation were identical to those previously determined with the conventional assay (k cat ϭ 3.2 s Ϫ1 ; K m (tRNA) ϭ 0.31 M), verifying that the new experimental approach is suitable for quantitative kinetic studies (13,15). The crystal structure of the GlnRS⅐tRNA Gln complex bound to glutamate and an ATP analog showed that the noncognate amino acid binds in a distinct orientation, in which the ␣-carboxylate is displaced by ϳ1 Å as compared with the position occupied by the equivalent moiety of cognate glutamine (6). This finding suggested that a significant component of amino acid discrimination resides at the chemical step of the reaction rather than in the initial binding. To evaluate this hypothesis, we examined noncognate aminoacylation by single-turnover kinetics, in which reactions were carried out with molar excess of enzyme over tRNA (Fig. 2). These noncognate reactions were sufficiently slow to allow sampling by hand, in contrast to cognate glutaminylation, which required rapid mixing (15). In GlnRS, formation of the activated aminoacyl adenylate intermediate requires the presence of tRNA (1), making isolation of the second tRNA transfer step difficult. Therefore, for these reactions we measured a composite k chem (Fig. 3), which represents the two-step reaction using ATP, amino acid, and tRNA Gln substrates and generating aminoacyl-tRNA Gln , AMP, and PP i .
The concentrations of ATP, glutamate, and tRNA Gln were systematically varied to determine conditions for substrate saturation. For cognate reactions performed in the rapid quench apparatus, systematic mixing controls previously established that the rate of product formation does not depend upon which substrates are preincubated with enzyme (15). Saturation for glutaminylation was established at 3 M tRNA Gln , 10 mM glutamine, and 10 mM ATP; under these conditions a single-exponential fit of the data yields a maximal k chem of 28 s Ϫ1 (15) ( Table II). This represents the rate of all reaction steps up to and including product formation on the enzyme. For the noncognate reaction saturation was established at 10 mM ATP and at 33 M tRNA Gln . However, titration of the glutamate concentration across a wide range showed that for this substrate the rate of product formation increases linearly up to 2 M without exhibiting saturation (Fig. 4A). Control experiments demonstrated that the k cat of cognate reactions is reduced by only 2-fold in the presence of 2.7 M NaCl; this effect is small, but may nonetheless limit detection of binding saturation at very high glutamate concentrations. Because saturation is not established, k chem for glutamylation determined at the highest glutamate concentration tested rep-resents a lower limit for the reaction chemistry (k chem Ͼ 0.17 s Ϫ1 ; Table II). Thus, GlnRS discriminates against glutamate at the chemical steps by at most 165-fold. The inability to detect glutamate saturation precludes determination of the glutamate K m parameter in the steady-state reaction (Table II; the k cat is determined with reference to tRNA Gln , see below).
The inability to saturate glutamate in the two-step aminoacylation is in contrast to the saturation that is observed in the ATP-PP i exchange assay, in which a K m value of 252 mM could be determined ( Fig. 4B and Table I). Possibly, this distinction is due to the significantly lower pH at which the ATP-PP i reaction has been performed (pH 6.0 versus pH 7.3 in the two-step aminoacylation). Although only a small fraction of glutamate is expected to be protonated at pH 6.0, protonation may be favored in the environment of the active site. Protonated glutamate would better resemble glutamine and would be expected to bind better than the negatively charged species (see "Discussion").
The dependence of the single-turnover reaction rate on the concentration of amino acid, in the presence of saturating levels of the other substrates, allows determination of the amino acid binding affinity by a hyperbolic fit of k chem with concentration. For cognate glutamine, amino acid binding is in rapid equilibrium with the enzyme (15); under these conditions, the K d of 1.1 mM determined by this kinetic approach is equivalent to the equilibrium binding constant (22). Because no saturation is observed for glutamylation, only a lower limit for the amino acid binding affinity can be established. From the observation that the reaction rate increases linearly up to 2 M glutamate ( Fig. 4), we roughly estimate K d (glutamate) to be above 750 mM. Thus, amino acid discrimination by GlnRS at the binding step may approach or exceed 10 3 -fold, so that specificity is apportioned somewhat more at the binding rather than the catalytic step. The concentration of glutamate in vivo is at the millimolar level, so the very weak affinity will certainly play the major role in precluding significant levels of Glu⅐tRNA Gln synthesis in the cell.
For cognate reactions, we previously showed that the ratelimiting step in the steady state is release of the Gln⅐tRNA Gln product. This is established by the observation of a rapid burst of Gln⅐tRNA Gln synthesis followed by a slow steady-state rate (15) (Fig. 5). In the case of the noncognate reactions, however, no burst could be observed under conditions where the first turnover can be readily detected (Fig. 5b), although the possibility of a shallow burst at very early time points cannot be excluded. However, the much slower rate observed in singleturnover reactions for synthesis of Glu⅐tRNA Gln would be consistent with an early rate-limiting step in the reaction pathway (Table II).
tRNA Gln Binding Affinity Determined by Transient Kinetics-The dependence of the single-turnover reaction rate on tRNA concentration was measured as a means of determining the affinity for tRNA under conditions of cognate aminoacylation. In these experiments, glutamine and ATP were kept at saturating levels and GlnRS was maintained in molar excess over tRNA. The appearance of the Gln⅐tRNA Gln product followed single-exponential kinetics at high concentrations of the complex (Fig. 6). However, at GlnRS concentrations between 0.24 M and 0.04 M a clear lag in product formation is observed. At yet lower concentrations (0.5-10 nM) the lag disappears, and the appearance of Gln⅐tRNA Gln again follows a first-order exponential. This kinetic behavior indicates that tRNA Gln is not in rapid equilibrium with GlnRS; hence, the K d for tRNA cannot be determined directly from a replot of the rate of the chemical step versus substrate concentration (as was possible for glutamine binding (15)). At high concentrations of the complex, the association rate k on(tRNA) is fast (see Fig. 3), and the enzyme⅐tRNA complex accumulates rapidly. Thus, the observed rate constant derived from the single-exponential fit reflects the chemical steps (Equation 1; see "Experimental Procedures"). The lag observed at intermediate concentrations indicates that the accumulation of unreacted tRNA and the sub-sequent reaction occur at similar rates. Product formation under these conditions can be fit to a double-exponential function (Equation 2). Finally, at the lowest concentrations the association of enzyme and tRNA becomes the rate-limiting factor and the formation of Gln⅐tRNA Gln reflects primarily this step.
To derive microscopic rate constants from these data, singleturnover reactions exhibiting a distinct lag phase were subjected to a nonlinear least squares regression analysis (Fig. 7). In this global fitting approach, the simulations are done assuming a two-step reversible sequence of reactions (Fig. 3), where k Ϫchem (reverse of the chemical step) is negligible for the aminoacylation of tRNA (22,24). The global fitting considers all four reactions displayed in Fig. 7 simultaneously and is capable of converging to an accurate solution given initial unfixed (floating) values for k on , k off , and k chem . Estimates for these parameters were extracted from three independent sets of data, yielding k on(tRNA) ϭ 1.2 ϫ 10 8 M Ϫ1 s Ϫ1 , k off(tRNA) ϭ 43 s Ϫ1 , and k chem ϭ 34 s Ϫ1 . The derived k chem value is very similar to the experimental determination (15) ( Table II). The global-fit parameters give K d of 0.36 M for tRNA Gln by the ratio of k off to k on . This estimate for K d is similar to the steady-state K m for tRNA (Table II).
As an independent approach to establish whether these values of the microscopic rate constants adequately describe the reaction mechanism, we performed pulse-chase experiments. In some systems, pulse-chase experiments allow the direct calculation of k on and k off rates for the titrated substrate. Pulsechase experiments reveal important information about the actual mechanistic details in catalysis in both a qualitative and quantitative fashion (see below). The methodology for pulsechase is analogous to that of the single-turnover experiments described above, except that the quenching step is performed by addition of a large molar excess of unlabeled bulk tRNA enriched in tRNA Gln (21). This quenching procedure does not denature the enzyme, so that bound, unreacted tRNA partitions between dissociation and aminoacylation (25,26). By contrast, the denaturing quench blocks all further forward progress of the reaction.
In these experiments the concentration of labeled tRNA Gln transcript was kept constant at 13 nM, whereas the concentration of GlnRS was varied from 40 to 240 nM (Fig. 8). For each reaction, we found that product formation fits well to a singleexponential function. A replot of k obs versus enzyme concentra- The overall scheme (top) describes the full two-step aminoacylation reaction. To simplify the drawing, in this scheme k on and k off indicate the association and dissociation rate constants, respectively, for any of the substrates. k activation is the rate constant for synthesis of the aminoacyl adenylate; k cat for ATP-PP i exchange (Table I; Fig. 1) measures the reverse of this reaction. k transfer is the forward rate constant for the second step of aminoacylation in which the amino acid is transferred to tRNA. For the two-step cognate reaction by wild-type GlnRS, k cat corresponds to product release, as shown. In the simplified scheme (bottom), k chem is a composite rate constant for the two-step reaction (Fig. 2); k on and k off are determined from substrate titrations under single-turnover conditions (see text and "Experimental Procedures"). tion (Fig. 8, inset) yields a slope k obs /[E] ϭ 1.7 ϫ 10 8 M Ϫ1 s Ϫ1 . Because of partitioning between tRNA dissociation and the forward reaction (defined by the (k chem )/(k chem ϩ k off ) term in Equation 6; see "Experimental Procedures"), this slope sets a lower limit for the association rate k on(tRNA) . Using the estimates of k off(tRNA) and k chem derived from global fitting, the parameter (k chem )/(k chem ϩ k off ) is estimated at 0.44, yielding in turn an estimate of k on(tRNA) ϭ 3.8 ϫ 10 8 M Ϫ1 s Ϫ1 . This is within 3-fold of the rate determined by global fitting of the denaturingquench reactions. In principle, k on(tRNA) and k off(tRNA) can be determined from the pulse-chase data using the relation [P] obs / [P] ϱ ϭ (k chem )/(k chem ϩ k off ), where [P] obs is the amount of product formed at the reaction time point where the concentration of the GlnRS⅐tRNA Gln complex is maximal (25). However, simulations showed that maximal accumulation of the complex occurs very rapidly, within the dead-time of the rapid quench instrument. Thus, an accurate estimate of [P] obs could not be made.
The rate of association for tRNA Gln is at or very near to that expected if diffusion is controlling, indicating very rapid binding of the tRNA under conditions of the enzymatic reaction. k on is also roughly 10-fold higher than the second-order rate constant k cat /K m (15) (Table II). A similar discrepancy between k on and k cat /K m was also observed for the hydrolysis reaction catalyzed by the RNA component of RNase P (26), and may indicate the existence of a distinct, nonequilibrating enzyme species formed during the process of product dissociation and subsequent substrate rebinding. Determination of the tRNA off-rate in a binary complex with GlnRS by fluorescence previously yielded k off(tRNA) ϭ 7 s Ϫ1 , suggesting that the affinity for tRNA in the context of a catalytically competent quaternary complex is somewhat weakened relative to a binary complex (27). This is consistent with the need for rapid turnover in the steady state.
Amino Acid-dependent tRNA Binding Affinity-We next examined the dependence of tRNA binding on the identity of the bound amino acid. Steady-state kinetic measurements first showed that tRNA Gln binding does approach saturation for noncognate glutamylation reactions carried out at 2 M glutamate. The K m for tRNA Gln is determined to be 19 M, some 60-fold higher than its K m in the cognate reactions. Because the tRNA saturates, we could also determine an apparent k cat , which is decreased by only 70-fold compared with the cognate reaction.
In contrast with the cognate reactions, single-turnover glutamylation time courses using the denaturing quench methodology failed to reveal a detectable lag phase at any tRNA concentration (Fig. 9). It thus appears that for noncognate glutamylation the formation of the GlnRS⅐tRNA Gln complex is in rapid equilibrium (k off(tRNA) Ͼ Ͼ k chem ), allowing determina-  ) and k on (tRNA Gln ) are the average of values derived by allowing all variables to float in the global fitting, and values derived by fixing k on and k chem . Errors for these parameters were taken as the standard deviations of these estimates from the mean.
f The K d reported is estimated from global fitting. K d determined by directly replotting the data on a hyperbolic binding isotherm is 6.7 M (see Fig. 8) .   FIG. 4. a, dependence of reaction rates on amino acid concentration. Replots of amino acid concentration versus k chem for glutaminylation (panel a at left (15)) and glutamylation (panel b at right). k chem is equivalent to the composite rate constant k chem (see reaction scheme in "Experimental Procedures"). Glutamine is in rapid equilibrium with the GlnRS⅐tRNA Gln ⅐ATP complex, allowing determination of K d from a hyperbolic fit (Equation 4) of the curve (15) ( Table II). The replot of glutamate concentration versus k chem shows that the enzyme complex is not saturated at concentrations up to 2.0 M. b, saturation of glutamate binding at pH 6.0 in ATP-PP i exchange. Hyperbolic replot of k activation (see Fig. 3) versus glutamate concentration (r ϭ 0.985) demonstrating saturation at pH 6.0. tion of K d by fitting the plot of k obs versus enzyme concentration to a standard hyperbolic isotherm. This yields K d of 6.7 M for tRNA Gln (Fig. 10).
The data were also subjected to global fitting to estimate the values of the association (k on(tRNA) ) and dissociation rate constants (k off(tRNA) ) for tRNA Gln (Fig. 11). Nonlinear leastsquares regression analyses were again performed by floating all three of the variables k on , k off , and k chem , yielding dissociation rate (k off(tRNA) ) of 60 s Ϫ1 , similar to the dissociation rate for the cognate reaction (Table II). However, the derived association rate of 4.5 ϫ 10 6 M Ϫ1 s Ϫ1 is ϳ100-fold lower than for the cognate reaction, suggesting that structural rearrangements upon tRNA binding may be perturbed in the formation of the noncognate quaternary complex. As observed for the cognate reactions, the derived value of k chem is again similar to that measured experimentally, although some increased uncertainty exists here due to the lack of glutamate saturation (Table II; k chem Ͼ 0.17 s Ϫ1 ). The ratio of the association and dissociation rate constants yields K d for tRNA Gln of 12.3 M. This represents 60-fold weaker affinity for tRNA than in the cognate reaction, and is within 2-fold of the value determined in the hyperbolic replot.
To further examine noncognate glutamylation, we again performed single-turnover reactions using a cold-quench solution containing excess tRNA (Fig. 12). Application of this methodology to the cognate reactions had revealed that the reaction rates under equivalent conditions were very much faster for the cold-quenching as compared with the denaturing quench protocol. For example, at the 10-ms time point, the cold-quench cognate reaction has reached 25% product formation, whereas the denaturing-quench cognate reaction shows only ϳ2.5% product formation. In sharp contrast, for the noncognate glutamylation we observed almost no difference in the extents of product formation for equivalent pulse-chase versus denaturing quench reactions (Fig. 13).
The different behavior observed in the noncognate reactions may be explained with reference to the replot of observed rates in the pulse-chase reactions, versus enzyme concentration (Fig.  12, inset). This replot yields a slope, k obs /[E] ϭ (k on )*(k chem )/ (k chem ϩ k off ), of 1.5 ϫ 10 4 M Ϫ1 s Ϫ1 , which sets a lower limit for the tRNA association rate. In contrast to the cognate reaction, where bound tRNA has roughly equal probability of partitioning back to dissociation or forward to reaction, in this case the rate constant for dissociation estimated from global fitting is much greater than the rate constant for the forward aminoacylation reaction (Table II). The ratio (k chem )/(k chem ϩ k off ) estimated from the global fitting parameters is ϳ0.005; thus, the observed slope k obs /[E] in the pulse-chase reactions is some 100-fold lower than the association rate constant. The slow association in noncognate reactions as well as the favored partitioning toward dissociation result in very little buildup of the pre-reactive complex; hence, little distinction is observed between reactions performed by the denaturing quench versus the pulse-chase methodologies.
As a further control for the effects of the high ionic strength in these reactions performed at very high glutamate concentrations, we performed single-turnover reactions by the denaturing quench protocol at 4-fold lower glutamate concentrations. We find that the kinetic K d under these conditions is 9.8 M (data not shown), very similar to the value derived from the measurements at 2 M glutamate (Table II). Clearly, the significant effect on tRNA Gln affinity caused by the presence of glutamate is not a consequence of weakened interactions due to high ionic strength. Instead, weakened tRNA affinity in the presence of the noncognate amino acid represents an inherent additional mechanism to ensure cognate aminoacyl-tRNA synthesis by GlnRS. We are unaware of previous studies demonstrating that tRNA affinity by a tRNA synthetase depends upon the identity of the bound amino acid.
Clearly, the presence of noncognate glutamate substantially alters the interaction of GlnRS with tRNA Gln . The slow association rate in these reactions suggests that the tRNA⅐enzyme complex may undergo a slow conformational transition after initial docking, in the presence of the noncognate amino acid. Although the crystal structure of the GlnRS⅐tRNA Gln complex bound to glutamate does not reveal misorientation of the tRNA, it is possible that any rearrangement in the final bound state is small, and may be masked by the lattice forces that drive an orientation for the tRNA that is both productive for catalysis and also favorable for crystal formation (6,28). DISCUSSION Pre-steady-state Kinetics by Chemical Quench-In this work we describe the application of both the conventional ATP-PP i exchange assay, as well as a more novel chemical-quench methodology to examine the origins of amino acid discrimination in a tRNA synthetase by pre-steady-state kinetics (6,15,16). The most important new feature of the chemical-quench assay is the use of 32 P-labeled tRNA. This gives excellent sensitivity while also permitting the use of very high concentrations of unlabeled amino acid. This approach should be readily generalizable to all tRNA synthetases, and will likely require only minor modifications to optimize TLC separations for the different aminoacyl adenylates. In addition, this assay offers a convenient method for measuring aminoacylation kinetics toward nonstandard amino acid substrates, most of which are not commercially available in radiolabeled form.
We demonstrate now that the chemical-quench assay is suitable for the reliable determination of both steady-state and microscopic kinetic parameters for aminoacylation, including estimation of ligand binding affinities and tRNA association and dissociation rates. This greatly benefits structure-function analyses, because for many applications it may reduce or eliminate the need to implement distinct binding assays. Moreover, it is important to note that the estimates of ligand affinities by these kinetic techniques yield values that are directly relevant to the enzyme under catalytic conditions. All other approaches to determining ligand affinity require that the reaction be blocked, either by eliminating a required substrate or cofactor, or by performing the reaction under noncatalytic conditions such as lowered pH. In favorable circumstances, such as when a reliable fluorescence-based binding assay is available, comparisons of ligand affinities determined by kinetics and by an alternative technique that omits the other substrate(s) may offer insight into ligand-induced structural rearrangements in the active site. Such an approach may be of particular value in tRNA synthetases and other RNA-modifying enzymes, where induced-fit assembly of the active-site conformation upon ligand binding is a common phenomenon (9).
Amino Acid Specificity in GlnRS at the Activation Step-Crystal structures of GlnRS bound in quaternary complexes with tRNA Gln , the nonhydrolyzable ATP analog AMPCPP, and either glutamine or glutamate showed that the enzyme binds glutamate in a distinct nonproductive orientation (6). The nonproductive binding is driven by a new ionic contact with the guanidinium group of Arg-30 that is not made by glutamine. This predicts that a major component of the 10 7 -fold discrimination against glutamate occurs at the first step of aminoacylation, when the free amino acid binds.
To test this hypothesis we isolated the first step of the reaction using the conventional ATP-PP i exchange assay.
These data showed that the discrimination against glutamate is 4 ϫ 10 4 -fold (Table I), accounting for a major fraction of the overall 10 7 -fold specificity. In this reaction the discrimination against glutamate arises from at most a 500-fold decrease in k cat together with a 100-fold increase in K m (Table I). Although we have not measured the tRNA transfer step independently, it is possible that the rates with which Gln-AMP and Glu-AMP react with the 2Ј-OH group at A76 of the tRNA will differ less significantly. This is because binding of the activated Gln-AMP and Glu-AMP intermediates could be driven by the common AMP moiety, resulting in a similar orientation of the mixed anhydride linkage relative to the tRNA ribose. In support of this conjecture, a 3.0-Å crystal structure of the GlnRS⅐tRNA Gln complex bound to a nonhydrolyzable glutamol adenylate analog 2 shows that the AMP portion of the analog binds nearly identically to the AMP portion of the glutamine analog 5Ј-O-[N-(L-glutaminyl)sulfamoyl] adenosine (QSI) (7). The k cat for Glu-AMP synthesis (k cat ϭ 0.09 s Ϫ1 ) is also very similar to the k chem for the overall two-step reaction under the same conditions (k chem ϭ 0.06 s Ϫ1 ). 3 Thus, activation of glutamate may be rate-determining for the overall two-step reaction.
Human GlnRS also shows greater amino acid selectivity for overall aminoacylation as compared with the first activation step, although the difference in that case is only 4-fold instead of the 10 2 -to 10 3 -fold for E. coli GlnRS (29). This distinction arises from more efficient misaminoacylation by the human enzyme. In E. coli GlnRS, the in vitro discrimination factor as assessed from the relative k cat /K m values is 4 ϫ 10 4 -fold, which is substantially higher than the observed overall error rate in protein biosynthesis (ϳ3 in 10,000). However, intracellular concentrations of glutamate in Gram-negative bacteria are generally at least 10-fold higher than those of glutamine (30).
Amino Acid Discrimination at the Binding and Chemical Steps-The rapid chemical-quench approach to examine cognate aminoacylation by GlnRS has revealed that the ratelimiting step in the catalytic cycle corresponds to release of aminoacyl-tRNA (15). Thus, the steady-state constants k cat and K m are not equivalent to chemical catalysis and substrate binding, respectively. Because of this, an assessment of how amino acid specificity is partitioned at the binding and catalytic steps, and further correlations of the free-energy differences at these steps with crystal structures, necessarily requires the use of transient kinetics. To address this, we employed single-turnover experiments with enzyme in excess of tRNA, at saturating concentrations of ATP and tRNA. The maximal rate of the chemical steps (k chem ) is found at saturating levels of amino acid, whereas the binding affinity is derived (for the rapid equilibrium case) by plotting k obs versus amino acid concentration and fitting to a hyperbolic binding equation. These experiments showed that the glutamate binding site in GlnRS is not saturable even at concentrations up to several molar, so that partitioning of specificity between binding and catalysis can be set only within limits (Table II).
The extremely weak glutamate binding affinity is unexpected, because x-ray crystal structures of the GlnRS⅐tRNA Gln complex bound to an ATP analog and either glutamine or glutamate showed apparently stronger binding interactions for the noncognate substrate (6). The side chain of free glutamine binds in the identical position as compared with the enzyme⅐tRNA complex bound to QSI (6,7). In both the unliganded and aminoacyl adenylate states, the amide side-chain oxygen of glutamine makes no discriminating contacts with enzyme groups, whereas the NH 2 group interacts with the phenolic hydroxyl of Tyr-211 and with a water molecule, each of which are of ambiguous hydrogen-bonding character. By contrast, glutamate binds in a distinct orientation in which one carboxylate oxygen makes an ion-pair contact with the guanidinium moiety of Arg-30, whereas the other oxygen occupies the same position as does -NH 2 of glutamine. In this binding mode the ␣-carboxylate group of glutamate is displaced with respect to the ribose sugar of the 3Ј-terminal tRNA nucleotide A76. Consequently, it appeared that the rate of the chemical step might be strongly decreased owing to the misorientation of reactive moieties, whereas the binding affinity for glutamate might be strong due to the ion-pair with Arg-30. The relatively weak binding affinity of 1 mM for glutamine is consistent with the absence of strong, orienting polar contacts for the amide group (6).
Discrimination against glutamate at the binding step by GlnRS may arise in part from unfavorable moderate-range electrostatic forces acting to oppose the apparently strong Arg-30 ion-pair. This is suggested based on a comparative analysis of the amino acid binding pockets in GluRS and Gl-nRS, as revealed by x-ray crystallography and extensive sequence alignments (6,31,32). As closely related class Ib tRNA synthetases, GluRS and GlnRS possess topologically identical active-site domains, with the ATP, amino acid, and tRNA 3Јend binding sites in very similar relative orientations. In the structure of the Thermus thermophilus GluRS⅐tRNA Glu complex bound to glutamate, the substrate side-chain carboxylate group makes three direct ion-pair contacts with two arginine residues conserved among GluRS enzymes (Arg-5 and Arg-205), the first of which corresponds to the strictly conserved Arg-30 in GlnRS (31). Because all GlnRS enzymes lack the second arginine (present at the position corresponding to Cys-229 in E. coli GlnRS), the electrostatic potential in the amino acid cleft should be significantly more negative than for GluRS. This is likely a primary mechanism by which GlnRS excludes glutamate; indeed, mutation of the equivalent amino acid in human GlnRS generated a modest increase in the activity of the enzyme toward glutamate (29).
Comparison of ATP-PP i exchange and overall aminoacylation kinetics also gives some insight into the origins of discrimination against glutamate. At the lower pH of the ATP-PP i exchange assay (pH 6.0), saturation for glutamate was observed (Fig. 4B). This is in sharp contrast to steady-state and single-turnover experiments for the overall reaction at pH 7.2 or pH 7.4, where no saturation was found (Fig. 4A). This suggests that partial protonation of glutamate at lower pH may render the noncognate substrate more similar to glutamine in its chemical properties. However, the protonation of titratable amino acid side chains in the binding cleft, such as His-215 (Fig. 14) or Cys-229, may provide necessary electrostatic compensation needed for stronger glutamate binding.
Multiple sequence alignments of GlnRS sequences show an accumulation of negatively charged residues in the GlnRS catalytic domain near the amino acid binding cleft (9).