HEK293E Cells Research Articles

Different Glycosylation Pattern of Human IgG1 and IgG3 Antibodies Isolated from Transiently as well as Permanently Transfected Cell Lines

The effector functions of IgG depend on the presence of carbohydrates attached to asparagine 297 in the Fc-portion. In this report, glycosylation profiles of recombinant wild-type and mutant IgG1 and IgG3 antibodies produced from three cell lines were analysed using LC-ESI-Orbitrap. Clear differences were detected between IgG1 and IgG3 glycoforms, where IgG1 generally contained fucosylated glycoforms, whilst IgG3 mainly were non-fucosylated. When using NS-0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, whilst IgG3 wt glycoforms did not. Transiently transfected HEK 293E cells were used to produce IgG1 and IgG3 wt and mutants, affecting complement activation. Cell supernatants were harvested at early and late time points and analysed separately. IgGs harvested late showed simpler and less developed glycosylation structure compared to those harvested early. The IgG harvested early was slightly more effective in complement activation than those harvested late, whilst the antibody-dependent cell-mediated cytotoxicity was unaltered. Generally, the glycosylation pattern of the mutants tested, including a hinge truncate mutant of IgG3, did not differ significantly from the wild-type IgGs. The striking difference in glycosylation pattern of IgG1 compared to IgG3 therefore appears not to be due to the long hinge region of IgG3 (62 amino acids) relative to the IgG1 hinge region (15 amino acids). Furthermore, mutation variants at or near the C1q binding site showed similar glycosylation structure and difference in their complement activation activity observed earlier is thus most likely due to differences in protein structure only.

A new method to customize protein expression vectors for fast, efficient and background free parallel cloning.

BackgroundExpression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector.ResultsHere we describe a method to tailor selected expression vectors for parallel Sequence and Ligation Independent Cloning. SLIC cloning enables precise and sequence independent engineering and is based on joining vector and insert with 15–25 bp homologies on both DNA ends by homologous recombination. We modified expression vectors based on pET, pFastBac and pTT backbones for parallel PCR-based cloning and screening in E.coli, insect cells and HEK293E cells, respectively. We introduced the toxic ccdB gene under control of a strong constitutive promoter for counterselection of insert less vector. In contrast to DpnI treatment commonly used to reduce vector background, ccdB used in our vector series is 100% efficient in killing parental vector carrying cells and reduces vector background to zero. In addition, the 3’ end of ccdB functions as a primer binding site common to all vectors. The second shared primer binding site is provided by a HRV 3C protease cleavage site located downstream of purification and solubility enhancing tags for tag removal. We have so far generated more than 30 different parallel expression vectors, and successfully cloned and expressed more than 250 genes with this vector series. There is no size restriction for gene insertion, clone efficiency is > 95% with clone numbers up to 200. The procedure is simple, fast, efficient and cost-effective. All expression vectors showed efficient expression of eGFP and different target proteins requested to be produced and purified at our Core Facility services.ConclusionThis new expression vector series allows efficient and cost-effective parallel cloning and thus screening of different protein constructs, tags and expression hosts.

An efficient strategy for cell-based antibody library selection using an integrated vector system

BackgroundCell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9.ResultsA mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9.ConclusionsThis study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.

Poly(ethyleneimine)‐Mediated Large‐Scale Transient Gene Expression: Influence of Molecular Weight, Polydispersity and N‐Propionyl Groups

Three synthesis lots of linear poly(ethyleneimine) (PEI) are compared to a fully hydrolyzed linear PEI (commercially available as PEI "Max") regarding structure, polyplex formation with plasmid DNA, and transfection of suspension-adapted HEK-293E cells. PEI "Max" binds DNA more efficiently than the other PEIs, but it is the least effective in terms of transient recombinant protein yield. One PEI lot is fractionated by means of SEC. The fractions of high-M(n) PEI are the most efficient for complex formation and transfection. Nevertheless, the highest transient recombinant protein yields are achieved with unfractionated PEI. The results demonstrate that the polydispersity and charge density of linear PEI are important parameters for gene delivery to suspension-adapted HEK-293E cells.

Glycan variability on a recombinant IgG antibody transiently produced in HEK-293E cells

In this study, a recombinant monoclonal IgG antibody was produced by transient gene expression (TGE) in suspension-adapted HEK-293E cells. The objective of the study was to determine the variation in recombinant IgG yield and glycosylation in ten independent transfections. In a ten-day batch process, the variation in transient IgG yield in the ten batches was less than 30% with the specific productivity averaging 20.2 ± 2.6 pg/cell/day. We characterized the N-glycosylation profile of each batch of affinity-purified IgG by intact protein and bottom-up mass spectrometry. Four major glycans were identified at Asn(297) in the ten batches with the maximum relative deviation for a single glycoform being 2.5%. In addition, within any single transfection there was little variation in glycoforms over the ten-day culture. Our experimental data indicate that with TGE, the production of recombinant IgG with little batch-to-batch variation in volumetric yield and protein glycosylation is feasible, even in a non-instrumented cultivation system as described here.

Reduced glutamine concentration improves protein production in growth-arrested CHO-DG44 and HEK-293E cells

For most cultivated mammalian cells, glutamine is an essential medium component. However, glutamine consumption results in the production of ammonia, a cytotoxic byproduct. Here we investigated the effect of glutamine reduction on recombinant protein production and ammonia accumulation in transiently transfected CHO and HEK-293E cells maintained under conditions of growth arrest. Maximum transient recombinant protein yields were observed in HEK-293E cultures without glutamine and in CHO cultures with 2 mM glutamine. The initial concentration of glutamine correlated with the level of ammonia accumulation in each culture. For both a stable CHO-derived cell line and a polyclonal population of recombinant CHO cells grown under conditions of mild hypothermia, the highest volumetric protein productivity was observed in cultures without glutamine. Here, the level of ammonia accumulation also corresponded to the initial glutamine concentration. Our data demonstrate that reduction of glutamine in the medium is an effective approach to improve protein production in both transiently and stably transfected mammalian cells when applying conditions that reduce or arrest the growth of these cells.

Influence of glutamine on transient and stable recombinant protein production in CHO and HEK-293 cells

BackgroundGlutamine is an essential component in culture mediafor most of the mammalian cell lines. It is often used asanalternativesourceofenergybycells,alongwithglu-cose. Glutamine metabolism induces ammonia accumu-lation in cell culture. Elevated ammonia concentrationabove 2 mM has been shown to have negative impacton both cell growth and recombinant protein productiv-ity [1-4]. In this study we investigated the effects ofdecreased glutamine concentration in the medium forCHO-DG44 and HEK-293E cells during transient geneexpression (TGE). The rationale was to reduce ammoniaaccumulation in the culture, and consequently, improvecell viability and recombinant protein productivity.Materials and methods

The use of filler DNA for improved transfection and reduced DNA needs in transient gene expression with CHO and HEK cells

Background Transient gene expression (TGE) is a rapid method for the production of recombinant proteins. Protein productivity in TGE has improved significantly over the past decade, reaching 300 mg/L and 1 g/L in CHO DG44 (CHO) and HEK 293E (HEK) cells, respectively [1,2]. However, the amount of plasmid DNA needed for transfection remains relatively high, contributing significantly to the overall cost of the TGE process. In order to reduce the amount of plasmid DNA in TGE, we examined the possibility of partially replacing it with herring sperm DNA (non-coding “filler” DNA) in transfections of CHO and HEK cells.

Pharmacological and Genetic Evaluation of Proposed Roles of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK), Extracellular Signal-regulated Kinase (ERK), and p90RSK in the Control of mTORC1 Protein Signaling by Phorbol Esters

Pharmacological and Genetic Evaluation of Proposed Roles of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK), Extracellular Signal-regulated Kinase (ERK), and p90RSK in the Control of mTORC1 Protein Signaling by Phorbol Esters

Functional reconstitution into liposomes of purified human RhCG ammonia channel.

BackgroundRh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH3, human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties.Methodology/Principal FindingsAn HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C12E8 detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH3 transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy.Conclusions/SignificanceThis study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent PunitNH3 (around 1×10−3 µm3.s−1) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60×10−3 µm3.s−1), and in human red blood cells endogenously expressing RhAG (2.18×10−3 µm3.s−1). The major finding of this study is that RhCG protein is active as an NH3 channel and that this function does not require any protein partner.

Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6

TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.

Enhancement of recombinant antibody production in HEK 293E cells by WPRE

In an effort to make a fast and convenient approach for efficient production of recombinant antibody, transient gene expression was performed in human embryonic kidney 293E (HEK293E) cells, which have been widely used as a mammalian host for transient expression of recombinant proteins. Woodchuck hepatitis virus post-transcriptional regulation element (WPRE) was employed to increase the antibody production. Under the influence of WPRE, the antibody production was increased by 5.5-fold through the enhancement of total mRNA levels of HC and LC, and the efficient export of nuclear mRNA into the cytoplasm. Using WPRE, 1.9 mg of cumulative recombinant antibody was obtained in transiently transfected adherent HEK293E cells from one 100 mm dish transfection with 10 mL medium exchange every 3 days for 24 days of cultivation. In addition, the highest recombinant antibody concentration of 81 mg/L was obtained. This simple and efficient approach of antibody production is expected to provide a sufficient amount of antibody for screening experiments.

Functional analysis of human RhCG: comparison with E. coli ammonium transporter reveals similarities in the pore and differences in the vestibule

Rh glycoproteins are members of the ammonium transporter (Amt)/methylamine permease (Mep)/Rh family facilitating movement of NH(3) across plasma membranes. Homology models constructed on the basis of the experimental structures of Escherichia coli AmtB and Nitrosomonas europaea Rh50 indicated a channel structure for human RhA (RhAG), RhB (RhBG), and RhC (RhCG) glycoproteins in which external and internal vestibules are linked by a pore containing two strictly conserved histidines. The pore entry is constricted by two highly conserved phenylalanines, "twin-Phe." In this study, RhCG function was investigated by stopped-flow spectrofluorometry measuring kinetic pH variations in HEK293E cells in the presence of an ammonium gradient. The apparent unitary NH(3) permeability of RhCG was determined and was found to be close to that of AmtB. With a site-directed mutagenesis approach, critical residues involved in Rh NH(3) channel activity were highlighted. In the external vestibule, the importance of both the charge and the conformation of the conserved aspartic acid was shown. In contrast to AmtB, individual mutations of each phenylalanine of the twin-Phe impaired the function while the removal of both resulted in recovery of the transport activity. The impact of the mutations suggests that, although having a common function and a similar channel structure, bacterial AmtB and human Rh vary in several aspects of the NH(3) transport mechanisms.

P90 Ribosomal S6 Kinase and p70 Ribosomal S6 Kinase Link Phosphorylation of the Eukaryotic Chaperonin Containing TCP-1 to Growth Factor, Insulin, and Nutrient Signaling

Chaperonin containing TCP-1 (CCT) is a large multisubunit complex that mediates protein folding in eukaryotic cells. CCT participates in the folding of newly synthesized polypeptides, including actin, tubulin, and several cell cycle regulators; therefore, CCT plays an important role in cytoskeletal organization and cell division. Here we identify the chaperonin CCT as a novel physiological substrate for p90 ribosomal S6 kinase (RSK) and p70 ribosomal S6 kinase (S6K). RSK phosphorylates the beta subunit of CCT in response to tumor promoters or growth factors that activate the Ras-mitogen-activated protein kinase (MAPK) pathway. CCTbeta Ser-260 was identified as the RSK site by mass spectrometry and confirmed by site-directed mutagenesis. RSK-dependent Ser-260 phosphorylation was sensitive to the MEK inhibitor UO126 and the RSK inhibitor BID-1870. Insulin weakly activates RSK but strongly activates the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway and utilizes S6K to regulate CCTbeta phosphorylation. Thus, the Ras-MAPK and PI3K-mTOR pathways converge on CCTbeta Ser-260 phosphorylation in response to multiple agonists in various mammalian cells. We also show that RNA interference-mediated knockdown of endogenous CCTbeta causes impaired cell proliferation that can be rescued with ectopically expressed murine CCTbeta wild-type or phosphomimetic mutant S260D, but not the phosphorylation-deficient mutant S260A. Although the molecular mechanism of CCTbeta regulation remains unclear, our findings demonstrate a link between oncogene and growth factor signaling and chaperonin CCT-mediated cellular activities.

Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells

Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient gene expression could offer a significant improvement, transient transfection is currently still limited by low titers and low specific productivity compared to stable cell lines. To overcome these bottlenecks, we have explored the use of various growth factors to enhance specific productivity and titers in the context of transient gene expression. For that purpose, several growth factors were cloned and screened for their effect on transient gene expression in HEK293E and CHO-DG44 cells. In particular, acidic fibroblast growth factor (aFGF) was able to increase specific productivity by 60% and recombinant protein titers by 80% in HEK293E cells, while FGF9 increased titers by 250% in CHO-DG44 cells.

Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology.

Regulation of Proline-rich Akt Substrate of 40 kDa (PRAS40) Function by Mammalian Target of Rapamycin Complex 1 (mTORC1)-mediated Phosphorylation

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [(32)P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition.

Crystal structure of Mabinlin II: A novel structural type of sweet proteins and the main structural basis for its sweetness

The crystal structure of a sweet protein Mabinlin II (Mab II) isolated from the mature seeds of Capparis masaikai Levl. grown in Southern China has been determined at 1.7A resolution by the SIRAS method. The Mab II 3D structure features in an "all alpha" fold mode consisting of A- and B-chains crosslinked by four disulfide bridges, which is distinct from all known sweet protein structures. The Mabinlin II molecule shows an amphiphilic surface, a cationic face (Face A) and a neutral face (Face B). A unique structural motif consisting of B54-B64 was found in Face B, which adopts a special sequence, NL-P-NI-C-NI-P-NI, featuring four [Asn-Leu/Ile] units connected by three conformational-constrained residues, thus is called the [NL/I] tetralet motif. The experiments for testing the possible interactions of separated A-chain and B-chain and the native Mabinlin II to the sweet-taste receptor were performed through the calcium imaging experiments with the HEK293E cells coexpressed hT1R2/T1R3. The result shows that hT1R2/T1R3 responds to both the integrated Mabinlin II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the sweetness alone, but A-chain does not. All data in combination revealed that the sweet protein Mabinlin II can interact with the sweet-taste receptor hT1R2/T1R3 to elicit its sweet taste, and the B-chain with a unique [NL/I] tetralet motif is the essential structural element for the interaction with sweet-taste receptor to elicit the sweetness, while the A-chain may play a role in gaining a long aftertaste for the integrate Mabinlin II. The findings reported in this paper will be advantage for understanding the diversity of sweet proteins and engineering research for development of a unique sweetener for the food and agriculture based on the Mabinlin II structure as a native model.

Structural Determinants of the C-terminal Helix-Kink-Helix Motif Essential for Protein Stability and Survival Promoting Activity of DJ-1

Mutations in the PARK7 gene encoding DJ-1 cause autosomal recessive Parkinson disease. The most deleterious point mutation is the L166P substitution, which resides in a structure motif comprising two alpha-helices (G and H) separated by a kink. Here we subjected the C-terminal helix-kink-helix motif to systematic site-directed mutagenesis, introducing helix-incompatible proline residues as well as conservative substitutions into the helical interface. Furthermore, we generated deletion mutants lacking the H-helix, the kink, and the entire C terminus. When transfected into neural and nonneural cell lines, steady-state levels of G-helix breaking and kink deletion mutants were dramatically lower than wild-type DJ-1. The effects of H-helix breakers were comparably smaller, and the non-helix breaking mutants only slightly destabilized DJ-1. The decreased steady-state levels were due to accelerated protein degradation involving in part the proteasome. G-helix breaking DJ-1 mutations abolished dimer formation. These structural perturbations had functional consequences on the cytoprotective activities of DJ-1. The destabilizing mutations conferred reduced cytoprotection against H(2)O(2) in transiently retransfected DJ-1 knock-out mouse embryonic fibroblasts. The loss of survival promoting activity of the DJ-1 mutants with destabilizing C-terminal mutations correlated with impaired anti-apoptotic signaling. We found that wild-type, but not mutant DJ-1 facilitated the Akt pathway and simultaneously blocked the apoptosis signal-regulating kinase 1, with which DJ-1 interacted in a redox-dependent manner. Thus, the G-helix and kink are critical determinants of the C-terminal helix-kink-helix motif, which is absolutely required for stability and the regulation of survival-promoting redox signaling of the Parkinson disease-associated protein DJ-1.

PRAS40 Is an Insulin-Regulated Inhibitor of the mTORC1 Protein Kinase

The heterotrimeric mTORC1 protein kinase nucleates a signaling network that promotes cell growth in response to insulin and becomes constitutively active in cells missing the TSC1 or TSC2 tumor suppressors. Insulin stimulates the phosphorylation of S6K1, an mTORC1 substrate, but it is not known how mTORC1 kinase activity is regulated. We identify PRAS40 as a raptor-interacting protein that binds to mTORC1 in insulin-deprived cells and whose in vitro interaction with mTORC1 is disrupted by high salt concentrations. PRAS40 inhibits cell growth, S6K1 phosphorylation, and rheb-induced activation of the mTORC1 pathway, and in vitro it prevents the great increase in mTORC1 kinase activity induced by rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which prevents its inhibition of mTORC1 in cells and in vitro. We propose that the relative strengths of the rheb- and PRAS40-mediated inputs to mTORC1 set overall pathway activity and that insulin activates mTORC1 through the coordinated regulation of both.