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• W2012209111 abstract "Early insulin signaling events were examined in a novel cell-free assay utilizing subcellular fractions derived from 3T3-L1 adipocytes. The following cellular processes were observed in vitro in a manner dependent on insulin, time of incubation, and exogenous ATP: 1) autophosphorylation and activation of the insulin receptor; 2) tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1); 3) association of tyrosine-phosphorylated IRS-1 with phosphoinositide 3-kinase; 4) activation of the kinase Akt via its phosphorylation on Thr-308 and Ser-473; and 5) phosphorylation of glycogen synthase kinase-3 by activated Akt. The activation of Akt in vitro was abolished in the presence of the phosphoinositide 3-kinase inhibitor, wortmannin, thus recapitulating the most notable regulatory feature of Akt observed in vivo. Evidence is presented indicating that the critical spatial compartmentalization of signaling molecules necessary for efficient signal transduction is likely to be preserved in the cell-free system. Additionally, data are provided demonstrating that full Akt activation in this system is dependent on plasma membrane-associated IRS-1, cannot be mediated by robust cytosol-specific tyrosine phosphorylation of IRS-1, and occurs in the complete absence of detectable IRS-2 phosphorylation in the cytosol and plasma membrane. Early insulin signaling events were examined in a novel cell-free assay utilizing subcellular fractions derived from 3T3-L1 adipocytes. The following cellular processes were observed in vitro in a manner dependent on insulin, time of incubation, and exogenous ATP: 1) autophosphorylation and activation of the insulin receptor; 2) tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1); 3) association of tyrosine-phosphorylated IRS-1 with phosphoinositide 3-kinase; 4) activation of the kinase Akt via its phosphorylation on Thr-308 and Ser-473; and 5) phosphorylation of glycogen synthase kinase-3 by activated Akt. The activation of Akt in vitro was abolished in the presence of the phosphoinositide 3-kinase inhibitor, wortmannin, thus recapitulating the most notable regulatory feature of Akt observed in vivo. Evidence is presented indicating that the critical spatial compartmentalization of signaling molecules necessary for efficient signal transduction is likely to be preserved in the cell-free system. Additionally, data are provided demonstrating that full Akt activation in this system is dependent on plasma membrane-associated IRS-1, cannot be mediated by robust cytosol-specific tyrosine phosphorylation of IRS-1, and occurs in the complete absence of detectable IRS-2 phosphorylation in the cytosol and plasma membrane. Insulin initiates multiple signaling pathways leading to numerous responses that regulate carbohydrate, fat, and protein metabolism (1Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Crossref PubMed Scopus (3813) Google Scholar). Hormone binding induces a conformational change in the insulin receptor that activates its intrinsic tyrosine kinase through an autophosphorylation mechanism. The activated receptor can then phosphorylate several intracellular protein substrates, most notably the insulin receptor substrate (IRS) 1The abbreviations used are: IRS, insulin receptor substrate; CYT, cytosol; GSK, glycogen synthase kinase; ins, insulin; LDM, low density microsomes; PI 3-kinase, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PM, plasma membrane; DTT, dithiothreitol; GST, glutathione S-transferase.1The abbreviations used are: IRS, insulin receptor substrate; CYT, cytosol; GSK, glycogen synthase kinase; ins, insulin; LDM, low density microsomes; PI 3-kinase, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PM, plasma membrane; DTT, dithiothreitol; GST, glutathione S-transferase. proteins (2White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 3White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (622) Google Scholar). Tyrosine-phosphorylated IRS proteins can recruit and activate the downstream effector, phosphoinositide 3-kinase (PI 3-kinase), which generates phosphatidylinositol 3,4,5-trisphosphate (PIP3) using inositol-containing phospholipids resident in the plasma membrane as substrates (4Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (829) Google Scholar). Many of the metabolic effects of insulin are absolutely dependent on PI 3-kinase activation. For example, insulin stimulation of glucose transport via translocation of the glucose transporter isoform Glut4 is completely blocked by the PI 3-kinase inhibitor wortmannin (5Clark J.F. Young P.W. Yonezawa K. Kasuga M. Holman G.D. Biochem. J. 1994; 300: 631-635Crossref PubMed Scopus (330) Google Scholar). In vitro assays have proven to be enormously useful for many areas of cell biology. The earliest studies, to our knowledge, in which this experimental approach was applied to the investigation of insulin action occurred during the late 1970s when L. Jarett and co-workers noted that the direct addition of insulin to a purified adipocyte plasma membrane fraction resulted in numerous effects, including alterations in the phosphorylation of several proteins (6Seals J.R. McDonald J.M. Jarett L. J. Biol. Chem. 1979; 254: 6991-6996Abstract Full Text PDF PubMed Google Scholar) and increased calcium binding by the plasma membrane (7McDonald J.M. Bruns D.E. Jarett L. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 1542-1546Crossref PubMed Scopus (61) Google Scholar). These investigators had very few guide-posts available at the time for interpreting their observations in a molecular context. Indeed, their work predated the cloning of the cDNA encoding the insulin receptor, which occurred in 1985 (8Ebina Y.E. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz F. Kan Y.W. Goldfine I.D. Roth R.A. Rutter W.J. Cell. 1985; 40: 747-758Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 9Ullrich A. Bell J.R. Chen E.Y. Herrera R. Petruzzelli L.M. Dull T.J. Gray A. Coussens L. Liao Y.C. Tsubokawa M. Mason A. Seeburg P.H. Grunfeld C. Rosen O.M. Ramachandran J. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1500) Google Scholar). More recently, the laboratory of C. R. Kahn employed subcellular fractions from 3T3-L1 adipocytes to reconstitute 1) the dynamic association of IRS-1/2 and PI 3-kinase with various cellular compartments (10Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) and 2) the binding of Glut4 vesicles to the plasma membrane (11Inoue G. Cheatham B. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14919-14924Crossref PubMed Scopus (15) Google Scholar). These investigators employed components derived from cells that were treated in vivo with or without insulin (100 nm for 10 min at 37 °C). The extent of manipulations that can be performed in their assay may, thus, be potentially limited due to the likelihood of the insulin-dependent process under investigation already having occurred in vivo before the time the components of their assay are recombined in vitro. This limitation may explain why the insulin-stimulated association of Glut4 vesicles with the plasma membrane that they observed in vitro was wortmannin-insensitive and did not require ATP or cytosol (11Inoue G. Cheatham B. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14919-14924Crossref PubMed Scopus (15) Google Scholar). During the current era in which complete genomic sequence data can be used in conjunction with sensitive proteomic techniques, it may be possible to comprehensively catalog all of the factors that are involved in bringing about the cellular responses elicited by insulin. As such information accrues, the challenge will increasingly shift toward untangling the web of functional interrelationships that exist among the identified factors. Efforts to devise in vitro assays that reconstitute cellular processes should be maintained to keep pace with the rate of discovery in insulin action. In this report we describe a novel in vitro assay reconstituting key aspects of PI 3-kinase-dependent insulin signaling using subcellular components isolated from 3T3-L1 adipocytes. Our data derived from this assay reinforce the functional significance of spatial compartmentalization exhibited by signaling components in the PI 3-kinase pathway. 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 (12Tordjman K.M. Leingang K.A. James D.E. Mueckler M.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7761-7765Crossref PubMed Scopus (115) Google Scholar). 3T3-L1 adipocytes were used 10–14 days after initiating differentiation. Isolation of Subcellular Components—Mature 3T3-L1 adipocytes grown on 10-cm dishes were serum-starved overnight. The cells were then rapidly washed 3 times with ice-cold serum-free Dulbecco's modified Eagle's medium and maintained further for 15 min at 4 °C in serum-free Dulbecco's modified Eagle's medium in the absence or presence of 1 μm insulin. Cells were then washed 3 times with ice-cold phosphate-buffered saline, scraped in 2 ml of ice-cold HES buffer (50 mm Hepes, pH 7.4, 255 mm sucrose, and 1 mm EDTA)/dish containing protease inhibitors (0.082 trypsin inhibitory units/ml aprotinin (Sigma), 1 μg/ml leupeptin, 1 μg/ml benzamidine, 1 μg/ml antipain, 5 μg/ml trypsin inhibitor, 1 μg/ml chymostatin, 1 μg/ml pepstatin A, and 0.5 mm phenylmethylsulfonyl fluoride), and then homogenized at 4 °C by passing the cells 10 times through a Yamato SC homogenizer at a speed of 1200 rpm. The plasma membrane (PM) fraction was obtained by differential centrifugation and sucrose cushion flotation as described previously (13Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar) and designated as either PM(–ins) or PM(+ins) according to whether the starting cell source was exposed to insulin. The low density microsomes (LDM) fraction was obtained from basal cells as described previously (13Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar). PM and LDM subsequent to their isolation were resuspended in IC buffer (20 mm Hepes, pH 7.4, 140 mm potassium glutamate, 5 mm NaCl, 1 mm EGTA, and protease inhibitors). A highly concentrated cytosol (CYT) fraction was prepared by washing the 3T3-L1 adipocytes 3 times with ice-cold IC buffer then removing the buffer as much as possible by aspiration followed by cell scraping and homogenizing with a ball-bearing homogenizer. The supernatant was recovered after an ultracentrifuge spin for 1 h at 200,000 × g. For the preparation of immunodepleted CYT, pre-cleared CYT was incubated for 1.5 h at 4 °C with protein A-Sepharose bound with the appropriate antibodies. A polyclonal rabbit antibody raised against the RNA-binding protein syncrip served as the control. In Vitro Assay—Samples were prepared on ice by mixing in various combinations LDM (∼2.5 mg/ml final concentration), CYT (∼3 mg/ml final concentration), and PM(+ or –ins) (∼0.5 mg/ml final concentration). Reaction volumes ranging from 100 to 200 μl were adjusted as necessary with IC buffer. Reactions were initiated with the addition of either an ATP regenerating system (final reaction concentrations, 1 mm ATP, 8 mm creatine phosphate, 30 units/ml creatine phosphokinase, and 5 mm MgCl2) or an ATP-depleting system (final reaction concentrations, 25 units/ml hexokinase and 5 mm glucose). Samples were rotated for 0–15 min in a 37 °C incubator. The reactions were quenched by the addition of an equal volume of buffer A (50 mm Hepes, pH 7.4, 150 mm NaCl, 2 mm sodium vanadate, 100 mm NaF, and 10 mm sodium pyrophosphate) either containing 2% SDS and 1 mm EDTA (for samples to be run directly on SDS-PAGE) or 2% Triton X-100 and 40 mm EDTA (for samples to be immunoprecipitated). For certain in vitro reactions, as indicated, some of the following were also added (final concentrations): 1 mm DTT, 150 μm sodium vanadate, 1 μm microcystin-LR (Calbiochem), 100 nm wortmannin (Calbiochem), or 1 μg/100 μl reaction volume of recombinant human insulin receptor cytoplasmic β subunit-GST fusion protein (Calbiochem; 407697). Immunoblot Analysis and Immunoprecipitation—Protein samples from the in vitro assay were subjected to SDS-PAGE and transferred to nitrocellulose. Phospho-specific antibodies recognizing the phosphorylated forms of Akt or glycogen synthase kinase-3 (GSK-3) were obtained from Cell Signaling Technology. The monoclonal anti-phosphotyrosine antibody PY20 was purchased from BD Biosciences. Immunoprecipitation and immunoblot analysis of IRS-1 was accomplished by use of a polyclonal rabbit IRS-1 antibody raised against the carboxy-terminal 14 amino acids of rat IRS-1. Insulin receptor, p85 subunit of PI 3-kinase, IRS-2, Akt1–3, and Gab1 antibodies were purchased from Upstate Biotechnology. The Akt3 antibody used for immunoblot analyses was from Santa Cruz Biotechnology. Characterization of the Cell-free System—We have attempted to reconstitute key aspects of the insulin-signaling pathway using subcellular fractions derived from 3T3-L1 adipocytes. We chose to use fully differentiated 3T3-L1 adipocytes as the source material because they are highly responsive to insulin. These cells typically exhibit a ∼10–20-fold increase in glucose uptake in response to acute stimulation with insulin (14Calderhead D.M. Kitagawa K. Tanner L.I. Holman G.D. Lienhard G.E. J. Biol. Chem. 1990; 265: 13801-13808Abstract Full Text PDF PubMed Google Scholar). The capacity to respond to this extent is acquired during the course of adipocyte differentiation, during which the expression levels of signaling components (such as the insulin receptor and IRS-1) (15Rubin C.S. Lai E. Rosen O.M. J. Biol. Chem. 1977; 252: 3554-3557Abstract Full Text PDF PubMed Google Scholar, 16Reed B.C. Kaufman S.H. Mackall J.C. Student A.K. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4876-4880Crossref PubMed Scopus (118) Google Scholar, 17Rice K.M. Lienhard G.E. Garner C.W. J. Biol. Chem. 1992; 267: 10163-10167Abstract Full Text PDF PubMed Google Scholar) and target molecules (such as the insulin-responsive glucose transporter Glut4) (12Tordjman K.M. Leingang K.A. James D.E. Mueckler M.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7761-7765Crossref PubMed Scopus (115) Google Scholar, 18James D.E. Strube M. Mueckler M. Nature. 1989; 338: 83-87Crossref PubMed Scopus (667) Google Scholar) are dramatically increased. Subcellular fractionation protocols exist for adipocytes that allow the reproducible recovery of distinct subcellular components with relative ease (13Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar, 19Jarett L. Methods Enzymol. 1974; 31: 60-71Crossref PubMed Scopus (81) Google Scholar, 20Simpson I.A. Yver D.R. Hissin P.J. Wardzala L.J. Karnielli E. Salans L.B. Cushman S.W. Biochim. Biophys. Acta. 1983; 763: 393-407Crossref PubMed Scopus (330) Google Scholar). An outline of our basic in vitro assay is provided in Fig. 1A. Fully differentiated 3T3-L1 adipocytes in the basal state were first cooled rapidly by washing with ice-cold media and then maintained at 4 °C in the presence or absence of 1 μm insulin. The cold temperature incubation allowed insulin to bind its cell surface receptor but prohibited subsequent intracellular signaling events. After the cold temperature incubation, purified PM fractions were obtained by differential centrifugation and sucrose cushion flotation. The PM fractions are referred to as PM(–ins) or PM(+ins) according to whether or not the cell source was exposed to insulin. Basal cells were also used to obtain the LDM and CYT. The LDM is enriched in endosomes, the Golgi apparatus, and insulin-responsive Glut4-containing vesicles as well as certain insulin-signaling molecules such as IRS-1 and PI 3-kinase (21Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar). In vitro reactions (∼100–200 μl) were prepared by mixing various combinations of the 3T3-L1 subcellular fractions (PM(–ins) or PM(+ins), LDM, or CYT) on ice. The reactions were initiated by the addition of an ATP regenerating system and then rotated for up to 15 min in a 37 °C incubator, thereby allowing insulin, carried through to this point in reactions containing PM(+ins) via high affinity interaction with its receptor, to exert its effects. The concentrations of PM, LDM, and CYT protein in a typical reaction were 0.5, 2.5, and 3 mg/ml, respectively. The delay in kinetics from what is observed in vivo is likely due in part to the time required for the temperature of the samples to rise from 4 to 37 °C and to the dilution of components in the cell-free system. The starting subcellular fractions were examined for the presence of insulin signaling molecules by immunoblot analysis (Fig. 1B). The insulin receptor was highly enriched in the PM fraction, and the amount did not vary with exposure to insulin. IRS-1 and IRS-2 were found mainly in the LDM and, to a lesser extent, the cytosol fraction, as previously reported (10Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). IRS-3, which is present in primary adipocytes, is not expressed in 3T3-L1 adipocytes (22Lavan B.E. Lienhard G.E. J. Biol. Chem. 1993; 268: 5921-5928Abstract Full Text PDF PubMed Google Scholar). In contrast to the IRS proteins, Gab1 (another insulin receptor substrate) (23Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (594) Google Scholar) was found exclusively in the cytosol. The p85 subunit of PI 3-kinase was present to a significant degree in all three subcellular fractions. Phosphoinositide-dependent kinase-1 was mainly found in the cytosol. Akt1–3 isoforms were present almost exclusively in the cytosolic fraction. We were particularly interested in reconstituting insulin-dependent processes downstream of PI 3-kinase. The generation of PIP3 by PI 3-kinase leads to the activation of Akt by phosphorylation of two of its residues, Thr-308 and Ser-473 (24Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2476) Google Scholar) (numerics corresponding to the Akt1 isoform). Akt in turn can phosphorylate GSK-3 on Ser-21 (for the α isoform) or Ser-9 (for the β isoform) (25Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4292) Google Scholar). The phosphorylation status of Akt and GSK-3 in our in vitro system was examined by immunoblot analysis using appropriate phospho-specific antibodies. The phospho-specific Akt antibodies used in this study (Cell Signaling Technology) are capable of detecting both Akt1 and Akt2 phosphorylation (26Hill M.M. Clark S.F. Tucker D.F. Birnbaum M.J. James D.E. Macaulay S.L. Mol. Cell. Biol. 1999; 19: 7771-7781Crossref PubMed Google Scholar), although Akt2 has been reported to be the major isoform in 3T3-L1 adipocytes (26Hill M.M. Clark S.F. Tucker D.F. Birnbaum M.J. James D.E. Macaulay S.L. Mol. Cell. Biol. 1999; 19: 7771-7781Crossref PubMed Google Scholar, 27Summers S.A. Whiteman E.L. Cho H. Lipfert L. Birnbaum M.J. J. Biol. Chem. 1999; 274: 23858-23867Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). As shown in Fig. 2, Akt was properly phosphorylated in vitro on both Thr-308 and Ser-473 in response to insulin in reactions containing PM (+ins), LDM, and CYT in the presence of 150 nm sodium vanadate and 1 μm microcystin-LR (lane 3). The occurrence of insulin-dependent signaling suggested that the cytoplasmic domain of the insulin receptor, which contains the intrinsic tyrosine kinase, was properly oriented with respect to the membrane to access its substrates in the cell-free system. This conforms to our estimate using alkaline phosphatase activity as an ecto-domain marker (28Garen A. Levinthal C. Biochim. Biophys. Acta. 1960; 38: 470-483Crossref PubMed Scopus (752) Google Scholar, 29Schlemmer S.R. Sirotnak F.M. J. Biol. Chem. 1992; 267: 14746-14752Abstract Full Text PDF PubMed Google Scholar), that 70% of the PM vesicles exhibited an inside-out orientation (data not shown). Moreover, the PM vesicles were apparently sealed, because Akt was not significantly activated by the direct addition of insulin to in vitro reactions containing PM(–ins) (lane 2). The addition of 100 nm wortmannin (lanes 4–6) or the absence of ATP (lanes 7–9) completely suppressed the insulin response. Reactions containing PM alone (lanes 10–12), CYT alone (lanes 13 and 14), and LDM alone (lanes 15 and 16) failed to result in insulin-stimulated phosphorylation of Akt. The absence of insulin-stimulated Akt phosphorylation in reactions containing PM, LDM, or CYT alone as well as the absolute requirement for exogenous ATP indicates that the system was not significantly contaminated by unbroken cells. Inhibition by wortmannin indicates that the insulin-stimulated Akt phosphorylation observed in vitro was PI 3-kinase-dependent. We attempted to optimize the conditions that elicited the maximal insulin response with regard to Akt and GSK-3 phosphorylation by performing the in vitro reactions in the absence or presence of phosphatase inhibitors (Fig. 3). Despite being enriched in signaling molecules such as PI 3-kinase and IRS-1/2 (21Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar), the LDM appeared to be dispensable for the phosphorylation of Akt. In fact, a stronger insulin-stimulated signal was consistently observed for both Akt phosphorylation sites in reactions excluding the LDM in the absence of the broad specificity Ser/Thr phosphatase inhibitor, microcystin-LR. This suggested that LDM might contain a phosphatase activity capable of acting on Akt. Generally, the tyrosine phosphatase inhibitor vanadate appeared to elevate the signal for both Akt phosphorylation sites in reactions containing PM(+ins). However, at the same time, the corresponding control (basal) signal in reactions containing PM(–ins) was also elevated, thus blunting the discernable insulin response. Microcystin also increased the insulin-stimulated signal for both Akt phosphorylation sites. However, this phosphatase inhibitor elevated the basal signal only for the Ser-473 site. Some of the in vitro reactions were also performed in the presence of 1 mm DTT, which was included to mimic the reducing environment found inside cells (Fig. 3; lower two rows). DTT appeared to inhibit the phospho-Akt signal in most cases. There are at least two reasons to explain why DTT failed to facilitate signaling. First, a reducing environment such as that provided by DTT is required for optimal activity of certain tyrosine phosphatases, which can be expected to down-regulate insulin signaling (30Takakura K. Beckman J.S. MacMillan-Crow L.A. Crow J.P. Arch. Biochem. Biophys. 1999; 369: 197-207Crossref PubMed Scopus (185) Google Scholar, 31Denu J.M. Tanner K.G. Biochemistry. 1998; 37: 5633-5642Crossref PubMed Scopus (814) Google Scholar). Second, DTT is known to inhibit the phosphatase-countering activity of vanadate (32Gordon J. Methods Enzymol. 1991; 201: 477-482Crossref PubMed Scopus (525) Google Scholar). The insulin-stimulated phosphorylation of GSK-3(α/β) on Ser-(21/9) mirrored that of Akt (Fig. 3). The empirical comparison of various conditions demonstrated that the optimal reaction for observing insulin-responsive phosphorylation of Akt and GSK-3 (i.e. the most dramatic fold difference between basal and insulin-stimulated signals) contained PM and CYT but excluded DTT, vanadate, and microcystin. Under these conditions both phosphorylation and dephosphorylation reactions could occur because phosphatase inhibitors were not necessary for detecting insulin-stimulated Akt phosphorylation. The time course for insulin receptor-mediated tyrosine phosphorylation under the optimal conditions described above was ascertained by immunoblot analysis using an anti-phosphotyrosine antibody. Two bands at ∼160 and ∼95 kDa appeared in response to insulin, corresponding to the molecular mass of IRS-1/2 and the β subunit of the insulin receptor, respectively (Fig. 4A). Phosphotyrosine signals were completely absent when ATP was depleted from the reaction (Fig. 4A, last four lanes), thus ruling out the possibility of significant reaction contamination by intact cells. The identities of the two insulin-dependent phosphotyrosine bands were confirmed by solubilizing the reaction with 1% Triton X-100 and then immunoprecipitating with an antibody recognizing either the β subunit of the insulin receptor or IRS-1 (Fig. 4B). For both of these proteins, the phosphotyrosine signal peaked at 2.5 min from the start of the reaction and somewhat decreased thereafter, presumably due to dephosphorylation. We also examined the in vitro recruitment of PI 3-kinase to tyrosine-phosphorylated adaptor proteins. After solubilizing the reaction with 1% Triton X-100, tyrosine-phosphorylated proteins capable of co-immunoprecipitating with the p85 subunit of PI 3-kinase were detected by immunoblot analysis (Fig. 4B, bottom panel). Insulin stimulated the association of PI 3-kinase with a tyrosine-phosphorylated protein doublet corresponding to the molecular mass of IRS-1 and IRS-2, mimicking what occurs in vivo (10Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 33Kelly K.L. Ruderman N.B. J. Biol. Chem. 1993; 268: 4391-4398Abstract Full Text PDF PubMed Google Scholar). A minor population of PI 3-kinase was found to be complexed with a protein of ∼95 kDa, which may be the autophosphorylated β subunit of the insulin receptor. It is important to note that others have observed an in vivo association between PI 3-kinase and the activated insulin receptor (34Endemann G. Yonezawa K. Roth R.A. J. Biol. Chem. 1990; 265: 396-400Abstract Full Text PDF PubMed Google Scholar, 35Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (389) Google Scholar). The time course for the phosphorylation of Akt was assessed by immunoblot analysis using phospho-Akt-specific antibodies (Fig. 5A). Akt phosphorylation exhibited kinetics delayed relative to that of IRS-1 tyrosine phosphorylation, with the insulin-stimulated signal peaking at ∼10–15 min from the start of the reaction. The time course for GSK-3 phosphorylation was similar to that of Akt (Fig. 5A). As was observed for tyrosine phosphorylation, the phosphorylation of Akt and GSK-3 in vitro was completely dependent on exogenous ATP (Fig. 5A, last four lanes). Also, the addition of 100 nm wortmannin to the reaction completely abrogated insulin-stimulated Akt phosphorylation on Thr-308 and Ser-473 and GSK-3(α/β) phosphorylation (Fig. 5B). This indicates that the in vitro kinase activities targeting both Akt sites and the subsequent GSK phosphorylation were PI 3-kinase-dependent, recapitulating in vivo characteristics (24Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2476) Google Scholar). Akt has been shown to translocate from the cytosol to the PM in response to insulin in intact cells (26Hill M.M. Clark S.F. Tucker D.F. Birnbaum M.J. James D.E. Macaulay S.L. Mol. Cell. Biol. 1999; 19: 7771-7781Crossref PubMed Google Scholar). In the basal state, Akt1–3 isoforms were found almost exclusively in the cytosolic fraction in our system (Fig. 1B). It was possible, however, that by incubating cells on ice with insulin before isolating the subcellular fractions, the system was “primed,” causing a residual amount of Akt1–3 to translocate to the PM and be phosphorylated after the addition of ATP. By immunodepleting Akt from the cytosol, it could be determined which form of Akt, cytosolic or PM-associated, was phosphorylated in the cell-free assay. As shown in Fig. 6A, each of the Akt isoforms could be collectively immunodepleted from the cytosol. In vitro reactions containing various combinations of PM(+ or –ins), CYT, and CYT(-Akt) revealed that insulin-dependent Akt and GSK-3 phosphorylation required the presence of both PM and CYT (Fig. 6B). No detectable Akt or GSK-3 phosphorylation was observed with PM alone. Immunodepleting Akt1–3 from the cytosol almost completely blocked insulin-dependent Akt and GSK-3 phosphorylation. These results indicate that Akt phosphorylated during the cell-free incubation originated from the cytosolic fraction, thus recapitulating what appears to occur in vivo. Insulin-stimulated Akt Activation Does Not Require Cytosolic IRS Proteins in the Cell-free Assay—The preceding data demonstrate that early insulin signaling events dependent on PI 3-kinase up to and including Akt and GSK-3 phosphorylation appear to be faithfully reconstituted in the in vitro system. A cell-free system offers several advantages that would be extremely difficult or impossible to address in a satisfactory manner using an intact cell system. In particular, facile experimental access to all components of our system allows manipulations such as the introduction of membrane-impermeant reagents or the depletion of cellular factors, as was illustrated in Fig. 6 with Akt. As a further demonstration of this principle, we added a soluble recombinant insulin receptor kinase domain fusion prote" @default.
• W2012209111 created "2016-06-24" @default.
• W2012209111 creator A5054724495 @default.
• W2012209111 creator A5075900186 @default.
• W2012209111 creator A5079562644 @default.
• W2012209111 date "2003-06-01" @default.
• W2012209111 modified "2023-09-26" @default.
• W2012209111 title "Reconstitution of Phosphoinositide 3-Kinase-dependent Insulin Signaling in a Cell-free System" @default.
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