Reconstitution of Phosphoinositide 3-Kinase-dependent Insulin Signaling in a Cell-free System

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 and maintained for min at in serum-free Dulbecco's in the absence or presence of insulin. were then washed 3 times with ice-cold in of ice-cold and inhibitor, and and then at by the cells 10 times through a at a of The plasma membrane fraction was obtained by and as described previously James D.E. J. PubMed Google Scholar) and as or to the cell was to insulin. The low density fraction was obtained from cells as described previously James D.E. J. PubMed Google Scholar). and to their were in and cytosol fraction was by the 3T3-L1 adipocytes 3 times with ice-cold then the as as possible by by cell and with a The was after an for h at For the of CYT, was for h at with protein with the the protein as the In were on by in various and or from to were as necessary with were with the addition of an ATP system and or an system and were for min in a 37 The were by the addition of an of and 10 and to be on or and to be For in vitro as of the following were DTT, nm wortmannin or of insulin receptor protein and from the in vitro assay were subjected to and to the of Akt or glycogen synthase kinase-3 were obtained from Cell The was from and of IRS-1 was by of a IRS-1 the of Insulin receptor, of PI 3-kinase, and were from The used for was from of the have to reconstitute key aspects of the using subcellular fractions derived from 3T3-L1 adipocytes. to 3T3-L1 adipocytes as the they are to insulin. These cells a in glucose in to stimulation with insulin K. Holman G.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The to to this extent is during the of adipocyte during which the of signaling components as the insulin receptor and E. Rosen O.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, K.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and molecules as the glucose transporter (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, D.E. M. Mueckler M. Nature. 1989; PubMed Scopus Google Scholar) are Subcellular exist for adipocytes that the of subcellular components with James D.E. J. PubMed Google Scholar, L. PubMed Scopus Google Scholar, E. PubMed Scopus (330) Google Scholar). of our in vitro assay is provided in 3T3-L1 adipocytes in the were rapidly by with ice-cold and then maintained at in the presence or absence of insulin. The insulin to its cell receptor intracellular signaling the incubation, purified fractions were obtained by and The fractions are to as or to or not the cell was to insulin. cells were used to the and The is in the and vesicles as as molecules such as IRS-1 and PI 3-kinase S. M.M. James D.E. J. Cell Biol. 1998; PubMed Scopus Google Scholar). In vitro were by various of the 3T3-L1 subcellular fractions or LDM, or on The were by the addition of an ATP system and then for to min in a 37 insulin, through to this in via with its receptor, to its The of PM, LDM, and protein in a were and 3 The in from is observed in vivo is likely due in to the time for the of the to from to 37 and to the of components in the cell-free system. The subcellular fractions were examined for the presence of insulin signaling molecules by The insulin receptor was in the and the did not with to insulin. IRS-1 and IRS-2 were in the to a the cytosol as previously (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). which is in is not in 3T3-L1 adipocytes J. Biol. Chem. Full Text PDF PubMed Google Scholar). In to the IRS insulin receptor M. Nature. PubMed Scopus Google Scholar) was in the The of PI 3-kinase was to a in all subcellular was in the were in the were in reconstituting insulin-dependent processes downstream of PI The of by PI 3-kinase to the activation of Akt by phosphorylation of of its Thr-308 and M. B. J. PubMed Scopus Google Scholar) to the Akt in can phosphorylate on the or the M. Nature. PubMed Scopus Google Scholar). The phosphorylation of Akt and in our in vitro system was examined by using The Akt used in this are of and phosphorylation M.M. James D.E. Mol. Cell. Biol. 1999; PubMed Google to be the isoform in 3T3-L1 adipocytes M.M. James D.E. Mol. Cell. Biol. 1999; PubMed Google Scholar, L. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). As in Akt was in vitro on Thr-308 and in to insulin in LDM, and in the presence of nm and The of insulin-dependent signaling that the of the insulin receptor, which the intrinsic tyrosine was with to the membrane to its substrates in the cell-free system. This to our using as an A. C. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google that of the vesicles exhibited an not the vesicles were Akt was not activated by the direct addition of insulin to in vitro The addition of nm wortmannin or the absence of ATP completely the insulin and and and to in insulin-stimulated phosphorylation of Akt. The absence of insulin-stimulated Akt phosphorylation in PM, LDM, or as as the for exogenous ATP that the system was not by by wortmannin that the insulin-stimulated Akt phosphorylation observed in vitro was PI to the that elicited the insulin with to Akt and phosphorylation by the in vitro in the absence or presence of in signaling molecules such as PI 3-kinase and IRS-1/2 S. M.M. James D.E. J. Cell Biol. 1998; PubMed Scopus Google the to be for the phosphorylation of Akt. In a insulin-stimulated signal was observed for Akt phosphorylation in the in the absence of the inhibitor, This that a of on Akt. the tyrosine inhibitor to the signal for Akt phosphorylation in at the the signal in was thus the insulin increased the insulin-stimulated signal for Akt phosphorylation this inhibitor the signal for the of the in vitro were performed in the presence of DTT, which was to the cells to the signal in most are at to explain why to a such as that provided by is for of tyrosine which can be to insulin signaling K. Biochem. 1999; PubMed Scopus Google Scholar, J.M. 1998; PubMed Scopus Google Scholar). is to the of J. PubMed Scopus Google Scholar). The insulin-stimulated phosphorylation of on that of Akt The of various that the for phosphorylation of Akt and the most and insulin-stimulated and DTT, and phosphorylation and were not necessary for insulin-stimulated Akt The time for insulin tyrosine phosphorylation under the described was by using an at and in to insulin, to the molecular of IRS-1/2 and the of the insulin receptor, were completely when ATP was from the thus the of by The of the insulin-dependent were by the with and then with an the of the insulin receptor or IRS-1 For of the signal at min from the of the and due to examined the in vitro of PI 3-kinase to tyrosine-phosphorylated the with tyrosine-phosphorylated proteins of with the of PI 3-kinase were by Insulin the association of PI 3-kinase with a tyrosine-phosphorylated protein to the molecular of IRS-1 and 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, J. Biol. Chem. Full Text PDF PubMed Google Scholar). of PI 3-kinase was to be with a protein of which may be the of the insulin is to that have observed an in vivo association PI 3-kinase and the activated insulin receptor G. Yonezawa K. Roth R.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. M.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The time for the phosphorylation of Akt was by using Akt phosphorylation exhibited to that of IRS-1 tyrosine with the insulin-stimulated signal at min from the of the The time for phosphorylation was to that of Akt As was observed for tyrosine the phosphorylation of Akt and in vitro was completely dependent on exogenous ATP the addition of nm wortmannin to the completely insulin-stimulated Akt phosphorylation on Thr-308 and and phosphorylation This that the in vitro kinase Akt and the phosphorylation were PI recapitulating in vivo M. B. J. PubMed Scopus Google Scholar). Akt to from the cytosol to the in to insulin in cells M.M. James D.E. Mol. Cell. Biol. 1999; PubMed Google Scholar). In the were in the fraction in our system was that by cells on with insulin before the subcellular the system was a of to to the and be after the addition of Akt from the it be which of or was in the cell-free As in of the Akt be from the In vitro various of or CYT, and that insulin-dependent Akt and phosphorylation the presence of and detectable Akt or phosphorylation was observed with from the cytosol completely blocked insulin-dependent Akt and These that Akt during the cell-free from the thus recapitulating to in vivo. Akt IRS in the data that insulin signaling events dependent on PI 3-kinase to and including Akt and phosphorylation to be in the in vitro system. cell-free system several that be or to in a manner using an cell system. In experimental to all components of our system manipulations such as the of or the of cellular as was in with Akt. As a of this we a insulin receptor kinase protein from the to an in vitro and The protein was tyrosine-phosphorylated in the absence of insulin, of its The signal derived from the insulin receptor protein was in to that derived from the insulin receptor in a and The insulin receptor protein was of IRS-1 The of tyrosine-phosphorylated IRS-1 was in the the insulin receptor protein as with the in which in vitro signaling was by insulin to our IRS-1/2 by the insulin receptor protein was in a with the regulatory of PI 3-kinase as by that the insulin receptor protein was on of substrate The tyrosine-phosphorylated insulin receptor protein was with a of the of of insulin the addition of the insulin receptor protein to activate downstream Thr-308 was In a and CYT, Akt was at regulatory The data that the signal from the insulin receptor at the plasma membrane in for Akt to be activated The activated insulin receptor can phosphorylate its substrates, the signaling at are likely to be and of downstream The most to signaling by the insulin receptor protein is at the of the activated PI 3-kinase in with IRS proteins may have limited to its phosphoinositide substrate in the of the plasma membrane. to signal In the signaling components are likely to be in such a as to be for action insulin are likely to be in an in vitro system in which the cellular components are by several of to the intracellular the PI 3-kinase-dependent Akt activation in the in vitro system is likely to the of signaling compartmentalization that in vivo at the the of the plasma membrane and the proteins be from signaling events using As in can be of IRS-1, or IRS-1 and IRS-2 in In vitro were performed using with the various In the and with an the of was observed by of the signal at in of IRS-1 from resulted in the absence of the of IRS-2 resulted in the absence of the This is with the of IRS-2 to As of IRS-1 and IRS-2 from resulted in the absence of the insulin-stimulated of from did not the of insulin-stimulated signaling to Akt was then by using The of proteins had on insulin-stimulated Akt Thr-308 and were in all of the This to that in and the that IRS proteins in the fraction are not for signaling to PI 3-kinase in the cell-free system. are several possible for IRS proteins may be for signaling to Akt. proteins may be for the PI 3-kinase necessary for Akt Akt signaling may be by a of PI 3-kinase to the activated insulin receptor in a manner to that for is that insulin activation of Akt may IRS with the In this it is notable that detectable of IRS-1, and are in the derived from our as in and the of IRS-1 and the of the that IRS proteins with the are for the signal that in Akt we performed a time for insulin-stimulated IRS-1 and IRS-2 tyrosine the of IRS proteins with the In vitro without cytosol were with the addition of at 37 and then after of time using an a of to the of the insulin receptor, that in to insulin and min after the of the before to the at 37 or for min at 37 without the addition of ATP exhibited tyrosine were (100 in with and then with IRS-1 or IRS-1 and IRS-2 were then for IRS-1 and IRS-2 were tyrosine-phosphorylated in an insulin-dependent manner that at min and or tyrosine phosphorylation was observed with isoform at min or at min at 37 in the absence of exogenous The that activated insulin receptor can tyrosine phosphorylate the of IRS-1/2 molecules with the in our in vitro we the exogenous insulin receptor protein phosphorylate In vitro in the absence or presence of were at 37 for were with IRS-1 or and then for In the absence of CYT, the insulin receptor not the exogenous insulin receptor tyrosine phosphorylate IRS-1 the addition of to the IRS-1 was tyrosine-phosphorylated by the activated receptor and the exogenous insulin receptor In IRS-2 was tyrosine-phosphorylated by the and insulin in the absence and presence of In the absence of a tyrosine phosphorylation was observed for IRS-2 in the insulin receptor with the activated receptor is not the phosphorylation is due to IRS-2 or tyrosine-phosphorylated proteins that with the in did not the of the exogenous insulin receptor with IRS-2 the following that insulin to Akt in the cell-free system. The signal from the insulin receptor at the plasma membrane in for Akt to be activated tyrosine phosphorylation of IRS-1, not to be the critical in the signaling leading to the of in the of the plasma membrane. phosphorylation of IRS-1/2 and their activation of PI 3-kinase to necessary for the stimulation of Akt The in at the to the phosphorylation of Akt on Thr-308 and by the James P.R. Biol. Full Text Full Text PDF PubMed Google Scholar, A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. M. A. R. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). aspects of our cell-free signaling are by several in vivo of a insulin receptor kinase D.E. M. Rosen O.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. Clauser E. Roth R.A. Rutter W.J. Mol. PubMed Scopus Google Scholar) not a resulted in in cells the that the was times in vitro L. Clauser E. Roth R.A. Rutter W.J. Mol. PubMed Scopus Google Scholar). The of IRS-1 as to IRS-2 in the activation of Akt is in with a that examined insulin signaling in cell derived from and IRS-2 M. J. K. K.M. M. M.F. Kahn C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). glucose was by in the IRS-2 cells with the PI 3-kinase was completely and the PI 3-kinase was in the IRS-2 adipocytes. In insulin activation of Akt as as the downstream of and kinase were by the absence of of investigation the of IRS compartmentalization in insulin For example, the of a IRS-1 to cell signaling through Akt insulin-stimulated tyrosine phosphorylation as as PI 3-kinase binding to IRS-1 M. J. K. K.M. M. M.F. Kahn C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of action for the IRS proteins is to in a manner the cytosol and intracellular (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, R.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the intracellular in 3T3-L1 adipocytes that IRS proteins with a the S. M.M. James D.E. J. Cell Biol. 1998; PubMed Scopus Google Scholar). that IRS-1/2 to the may be of to insulin signal by IRS proteins to be in to the insulin receptor at the S. M.M. James D.E. J. Cell Biol. 1998; PubMed Scopus Google Scholar, James D.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of IRS proteins from this subcellular to the cytosol in a of insulin-stimulated tyrosine phosphorylation of IRS-1/2 in 3T3-L1 adipocytes James D.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These observations completely the that not IRS are in insulin signal should all of the observed phosphorylation events occurred in including the autophosphorylation of the insulin receptor, we cannot the that such as the of a of IRS proteins to the PM, when cells were on with insulin before the of the subcellular that of 3T3-L1 adipocytes with insulin at in the autophosphorylation of the insulin receptor and the tyrosine phosphorylation of IRS-1, the of IRS-1 at the was not insulin-dependent R.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In our and cells in the of IRS proteins in the fraction were not this on of the presented the of insulin it on the of the cell-free assay to signaling In we have a novel cell-free assay reconstituting a PI 3-kinase-dependent signaling by insulin. The of in vivo PI 3-kinase to be activated by the receptor and to the intracellular the of the plasma membrane (4Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (829) Google Scholar). The of this dynamic process is to with from a our knowledge, this assay the in vitro of the of PI 3-kinase-dependent insulin from the of the insulin receptor activation. The experimental provided by this cell-free assay can be to the in