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• W2002398354 abstract "The ability of the insulin receptor to phosphorylate multiple substrates and their subcellular localization are two of the determinants that contribute to diversity of signaling. We find that insulin receptor substrate (IRS)-1 is 2-fold more concentrated in the intracellular membrane (IM) compartment than in cytosol, whereas IRS-2 is 2-fold more concentrated in cytosol than in IM. Insulin stimulation induces rapid tyrosine phosphorylation of both IRS-1 and IRS-2. This occurs mainly in the IM compartment, even though IRS-2 is located predominantly in cytosol. Furthermore, after insulin stimulation, both IRS-1 and IRS-2 translocate from IM to cytosol with at½ of 3.5 min. Using an in vitro reconstitution assay, we have demonstrated an association between IRS-1 and internal membranes and have shown that the dissociation of IRS-1 from IM is dependent on serine/threonine phosphorylation of IM. By comparison, within 1 min after insulin stimulation, 40% of the total pool of the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) is recruited from cytosol to IM, the greater part of which can be accounted for by binding to IRS-1 present in the IM. The p85 binding and phosphatidylinositol 3-kinase activity associated with IRS-2 rapidly decrease in both IM and cytosol, whereas those associated with IRS-1 stay at a relatively high level in IM and increase with time in cytosol despite a return of p85 to the cytosol and decreasing tyrosine phosphorylation of cytosolic IRS-1. These data indicate that IRS-1 and IRS-2 are differentially distributed in the cell and move from IM to cytosol following insulin stimulation. Insulin-stimulated IRS-1 and IRS-2 signaling occurs mainly in the IM and shows different kinetics; IRS-1-mediated signaling is more stable, whereas IRS-2-mediated signaling is more transient. These differences in substrate utilization and compartmentalization may contribute to the complexity and diversity of the insulin signaling network. The ability of the insulin receptor to phosphorylate multiple substrates and their subcellular localization are two of the determinants that contribute to diversity of signaling. We find that insulin receptor substrate (IRS)-1 is 2-fold more concentrated in the intracellular membrane (IM) compartment than in cytosol, whereas IRS-2 is 2-fold more concentrated in cytosol than in IM. Insulin stimulation induces rapid tyrosine phosphorylation of both IRS-1 and IRS-2. This occurs mainly in the IM compartment, even though IRS-2 is located predominantly in cytosol. Furthermore, after insulin stimulation, both IRS-1 and IRS-2 translocate from IM to cytosol with at½ of 3.5 min. Using an in vitro reconstitution assay, we have demonstrated an association between IRS-1 and internal membranes and have shown that the dissociation of IRS-1 from IM is dependent on serine/threonine phosphorylation of IM. By comparison, within 1 min after insulin stimulation, 40% of the total pool of the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) is recruited from cytosol to IM, the greater part of which can be accounted for by binding to IRS-1 present in the IM. The p85 binding and phosphatidylinositol 3-kinase activity associated with IRS-2 rapidly decrease in both IM and cytosol, whereas those associated with IRS-1 stay at a relatively high level in IM and increase with time in cytosol despite a return of p85 to the cytosol and decreasing tyrosine phosphorylation of cytosolic IRS-1. These data indicate that IRS-1 and IRS-2 are differentially distributed in the cell and move from IM to cytosol following insulin stimulation. Insulin-stimulated IRS-1 and IRS-2 signaling occurs mainly in the IM and shows different kinetics; IRS-1-mediated signaling is more stable, whereas IRS-2-mediated signaling is more transient. These differences in substrate utilization and compartmentalization may contribute to the complexity and diversity of the insulin signaling network. Insulin exerts a multifaceted and highly integrated series of actions via its intracellular signaling systems (1Kahn C.R. Diabetes. 1994; 43: 1066-1084Crossref PubMed Scopus (0) Google Scholar). The insulin receptor has intrinsic tyrosine kinase activity, which is activated upon insulin binding, leading to phosphorylation of the receptor and its substrates on tyrosine residues. Thus far, six substrates of the insulin receptor have been cloned, the best characterized of which are IRS-1 1The abbreviations used are: IRS, insulin receptor substrate; PI, phosphatidylinositol; IM, intracellular membrane; PM, plasma membrane; PH, pleckstrin homology; PTB, phosphotyrosine binding; PAGE, polyacrylamide gel electrophoresis; PP2A, protein phosphatase 2A. and IRS-2 (2Sun X.J. Rothenberg P.L. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1285) Google Scholar, 3Sun X.J. Wang L.M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E.M. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (765) Google Scholar, 4Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (601) Google Scholar, 5Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 6Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Following tyrosine phosphorylation, each of these intracellular substrates associates with SH2 domain-containing molecules to generate downstream signals (1Kahn C.R. Diabetes. 1994; 43: 1066-1084Crossref PubMed Scopus (0) Google Scholar). These SH2 domain molecules include phosphatidylinositol (PI) 3-kinase, Grb2, SHP2, fyn, nck, and crk (7Sun X.J. Pons S. Asano T. Myers Jr., M.G. Glasheen E.M. White M.F. J. Biol. Chem. 1996; 271: 10583-10587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 8Lee C.H. Li W. Nishimura R. Zhou M. Batzer A.G. Myers Jr., M.G. White M.F. Schlessinger J. Skolnik E.Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11713-11717Crossref PubMed Scopus (198) Google Scholar, 9Beitner-Johnson D. Blakesley V.A. Shen-Orr Z. Jimenez M. Stannard B. Wang L.-M. Pierce J.H. LeRoith D. J. Biol. Chem. 1996; 271: 9287-9290Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This complex array of signaling molecules not only participates in different parts of the insulin action cascade but also shows different distributions and patterns of movement within the cell. Insulin receptors are internalized following insulin stimulation, and the internalized receptors have a higher ability to phosphorylate substrates, such as IRS-1 (10Kublaoui B. Lee J. Pilch P.F. J. Biol. Chem. 1995; 270: 59-65Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 11Wang B. Balba Y. Knutson V.P. Biochem. Biophys. Res. Commun. 1996; 227: 27-34Crossref PubMed Scopus (17) Google Scholar). Previous studies have also shown that IRS-1 is present both on intracellular membranes and in cytosol (10Kublaoui B. Lee J. Pilch P.F. J. Biol. Chem. 1995; 270: 59-65Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 12Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Ricort J.M. Tanti J.F. Van Obberghen E. Le Marchand Brustel Y. Eur. J. Biochem. 1996; 239: 17-22Crossref PubMed Scopus (85) Google Scholar). In rat adipocytes and 3T3-L1 adipocytes, tyrosine phosphorylation of IRS-1 and association with PI 3-kinase occurs mainly in the intracellular membrane compartment (10Kublaoui B. Lee J. Pilch P.F. J. Biol. Chem. 1995; 270: 59-65Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 12Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Ricort J.M. Tanti J.F. Van Obberghen E. Le Marchand Brustel Y. Eur. J. Biochem. 1996; 239: 17-22Crossref PubMed Scopus (85) Google Scholar, 14Kelly K.L. Ruderman N.B. J. Biol. Chem. 1993; 268: 4391-4398Abstract Full Text PDF PubMed Google Scholar, 15Yang J. Clarke J.F. Ester C.J. Young P.W. Kasuga M. Holman G.D. Biochem. J. 1996; 313: 125-131Crossref PubMed Scopus (104) Google Scholar, 16Nave B.T. Haigh R.J. Hayward A.C. Siddle K. Shepherd P.R. Biochem. J. 1996; 318: 55-60Crossref PubMed Scopus (129) Google Scholar). The pleckstrin homology (17Myers Jr., M.G. Grammer T.C. Brooks J. Glasheen E.M. Wang L.-M. Sun X.J. Blenis J. Pierce J.H. White M.F. J. Biol. Chem. 1995; 270: 11715-11718Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 18Voliovitch H. Schindler D.G. Hadari Y.R. Taylor S.I. Accili D. Zick Y. J. Biol. Chem. 1995; 270: 18083-18087Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 19Yenush L. Makati K.J. Smith-Hall J. Ishibashi O. Myers Jr., M.G. White M.F. J. Biol. Chem. 1996; 271: 24300-24306Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) and the phosphotyrosine binding domains (20Eck M.J. Dhe-Paganon S. Trub T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) of IRS-1 are likely to enhance tyrosine phosphorylation of IRS-1 by the receptor and have been suggested to promote association of this substrate with the plasma membrane and the juxtamembrane region of the receptor. However, it is not clear why IRS-1 associates with internal membranes nor whether this feature of membrane association could be observed for other substrates. Likewise, little is known about intracellular trafficking of these molecules that might occur following insulin stimulation. Such movement and compartmentalization could play a role not only in activation of PI 3-kinase but also in activation of the Ras signaling pathway and the various serine/threonine kinases that are stimulated by insulin. In the present study, we have characterized the kinetics and differential subcellular localization of insulin signaling molecules, including IRS-1, IRS-2, and PI 3-kinase, in order to better understand the role of spatial distribution in insulin signaling, and we have studied the effects of serine/threonine phosphorylation on this process. Insulin was purchased from Boehringer Mannheim. Polyclonal anti-IRS-1 and anti-IRS-2 were prepared as described previously (21Bruning J.C. Winnay J. Cheatham B. Kahn C.R. Mol. Cell Biol. 1997; 17: 1513-1521Crossref PubMed Scopus (191) Google Scholar). Polyclonal antibodies to Ras and the p85 subunit of PI 3-kinase, monoclonal anti-phosphotyrosine antibody (4G10), and protein phosphatase type-2A (14-111) were purchased from Upstate Biotechnology Inc. (New York, NY). Polyclonal anti-Shc, anti-mSOS, and monoclonal anti-Grb2 were purchased from Transduction Laboratories. Anti-Akt was a gift of Dr. T. F. Franke. Anti-Gab-1 was a gift of Dr. D. Burks (Joslin Diabetes Center, Boston, MA). 3T3-L1 fibroblasts were maintained and differentiated using isobutylmethylxanthine, dexamethasone, and insulin as described previously (22Frost S.C. Lane M.D. J. Biol. Chem. 1985; 260: 2646-2652Abstract Full Text PDF PubMed Google Scholar). The adipocytes were used 10 days after the initiation of differentiation, by which time >90% of the cells had obtained an adipocyte morphology. Differentiated 3T3-L1 cells in 10-cm dishes were serum-starved for 18 h before experiments. For stimulation, cells were treated with 10−7m insulin for the indicated times, washed twice with ice-cold phosphate buffered saline, and immediately homogenized using 26 strokes of a 1-ml Teflon-glass homogenizer in a buffer containing 10 mm HEPES, pH 7.4, 1 mm EDTA, 255 mm sucrose, 1 mmNa3VO4, 50 nm okadaic acid, 1 mmphenylmethylsulfonyl fluoride, and 0.1 mg/ml aprotinin. In some experiments, 1 mm Na3VO4 and/or 50 nm okadaic acid were omitted from the homogenization buffer to allow dephosphorylation to occur. The homogenized cells were then subjected to subcellular fractionation as described previously to isolate plasma membranes, intracellular membranes, and cytosol (23Simpson I.A. Yver D.R. Hissin P.J. Wardzala L.J. Karnieli E. Salans L.B. Cushman S.W. Biochim. Biophys. Acta. 1983; 763: 393-407Crossref PubMed Scopus (330) Google Scholar, 24Clancy B.M. Czech M.P. J. Biol. Chem. 1990; 265: 12434-12443Abstract Full Text PDF PubMed Google Scholar). The protein concentration of these fractions was measured using the Bradford method. For immunoprecipitation, subcellular fractions were solubilized in the above-mentioned homogenization buffer supplemented with 1% (v/v) Nonidet P-40. After centrifugation, supernatants were normalized for protein concentration and subjected to immunoprecipitation. Lysates were incubated with primary antibody for 2 h at 4 °C and with protein A-Sepharose beads for additional 1.5 h, followed by three washes with lysis buffer before elution into SDS-PAGE sample buffer. The samples were then loaded onto a SDS-polyacrylamide gel and subjected to electrophoresis. Under the conditions used, our anti-IRS-1 precipitates about 90% of IRS-1 and virtually no IRS-2, whereas the anti-IRS-2 precipitates only about 37% of IRS-2 and about 7% of the IRS-1 (data not shown). For immunoblotting, equal amounts of protein (10–50 μg) were subjected to SDS-PAGE and electroblotted to nitrocellulose filters. The filters were blocked with 3% bovine serum albumin and then incubated with the appropriate antibody, washed, reacted with anti-rabbit or anti-mouse IgG coupled to peroxidase, and developed with enhanced chemiluminescence reagents as instructed by the manufacturer (NEN Life Science Products). The signals on the blot were quantified by densitometry. For PI 3-kinase assays, lysates of subcellular fractions were subjected to immunoprecipitation with anti-p85, anti-IRS-1, or anti-IRS-2 for 15 h followed by incubation with protein A-Sepharose for an additional 1.5 h at 4 °C. The immunoprecipitates were washed and subjected to the in vitro PI 3-kinase assay as described previously (25Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1001) Google Scholar). Intracellular membranes and cytosol were prepared from cells stimulated with or without 10−7m insulin for 10 min in homogenization buffer without or with phosphatase inhibitors (1 mmvanadate and/or 50 nm okadaic acid). The resulting pellet of the intracellular membranes was resuspended with cytosol from an equivalent number of either basal or insulin-stimulated cells prepared in homogenization buffer without or with phosphatase inhibitors. Each assay tube contained fractions from cells in one 10-cm dish in a total volume of 100 μl. The suspension was incubated for 30 min at 30 °C, after which intracellular membranes were reisolated by centrifugation at 200,000 × g for 1 h. IRS-1 in the resulting intracellular membranes was detected with immunoblotting with anti-IRS-1 antibody. In the study using protein phosphatase 2A, intracellular membranes were suspended in the homogenization buffer without phosphatase inhibitors, supplemented with 10 mmMgCl2, and then incubated with protein phosphatase 2A at 30 °C for 30 min. After the incubation, cytosol from an equivalent number of cells was added to the mixture, and the mixture was incubated at 30 °C for a further 30 min, followed by centrifugation to reisolate the intracellular membranes. Fig. 1 Ashows the subcellular distribution of tyrosine-phosphorylated proteins induced by insulin stimulation in 3T3-L1 adipocytes as determined by subcellular fractionation and immunoblotting with anti-phosphotyrosine. As described previously, tyrosine phosphorylation of the β-subunit of the insulin receptor (95 kDa) was maximally induced 1 min after insulin stimulation in the plasma membrane (PM) and slightly decreased during the 10-min time course. A trace of the phosphorylated insulin receptor was also observed in the intracellular membrane (IM). Whether this represents internalized insulin receptor or a small contamination of the IM with PM is unclear. The tyrosine-phosphorylated 52-kDa isoform of Shc was also recovered in the PM. Under the conditions of this experiment and in 3T3-L1 cells, the phosphorylation of this Shc isoform was observed in the basal state and was increased by about 30% following stimulation with insulin. Tyrosine-phosphorylated IRS-1 and IRS-2 migrate as a broad band at ∼185 kDa and were apparent in the IM and cytosol, but not in the PM. By scanning densitometry, insulin-induced phosphorylation of IRS-1/2 was 3-fold higher in the IM than in cytosol when normalized for protein amount, reached a peak at 1 min, and decreased thereafter with time in both the fractions (Fig.1 B). The level at which other insulin receptor substrates are present in 3T3-L1 cells is not known; however, a small amount of tyrosine-phosphorylated protein was observed in the PM at 60 kDa, which may represent IRS-3 (5Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 26Hosomi Y. Shii K. Ogawa W. Matsuba H. Yoshida M. Okada Y. Yokono K. Kasuga M. Baba S. Roth R.A. J. Biol. Chem. 1994; 269: 11498-11502Abstract Full Text PDF PubMed Google Scholar). To further analyze the insulin-stimulated tyrosine phosphorylation of pp185, both cytosol and IM were subjected to immunoprecipitation with anti-IRS-1 or anti-IRS-2 followed by immunoblot analysis of the immunoprecipitates with anti-phosphotyrosine. In agreement with Sunet al. (27Sun X.J. Pons S. Wang L.M. Zhang Y. Yenush L. Burks D. Myers Jr., M.G. Glasheen E. Copeland N.G. Jenkins N.A. Pierce J.H. White M.F. Mol. Endocrinol. 1997; 11: 251-262Crossref PubMed Scopus (123) Google Scholar), anti-IRS-1 precipitated over 90% of the total pp185 band, indicating that in 3T3-L1 cells IRS-1 is the predominate substrate of the insulin receptor (data not shown). Tyrosine phosphorylation of IRS-1 was maximal in IM within 1 min of insulin stimulation and declined slowly over 10 min (Fig. 1 C). IRS-2 phosphorylation in the IM also peaked at 1 min after stimulation, but in contrast to IRS-1, IRS-2 phosphorylation declined rapidly. In the cytosol, phosphorylation of both IRS-1 and IRS-2 was rapid but transient, peaking at 1 min and then declining to near basal levels by 4 min. Maximal phosphorylation of these two proteins in the cytosol was much lower, being 30 and 10% of the IM level for IRS-1 and IRS-2, respectively. Because IRS-1 contributes to most of the tyrosine-phosphorylation of pp185, we could calculate the apparent stoichiometry of IRS-1 tyrosine phosphorylation in individual fractions at each time point (Fig. 1 E). Despite the relative abundance of IRS-1 in the IM, the stoichiometry of phosphorylation was similar in both the IM and cytosol at 1 min after insulin stimulation. Interestingly, in the IM, the stoichiometry continued to increase with time, whereas that in cytosol declined by more than 50%. This suggests that tyrosine-phosphorylated IRS-1 in IM dissociates to cytosol and is then dephosphorylated, rather than both effects occurring in both compartments. To determine whether the compartmentalization of phosphorylation was due to a difference in substrate protein localization or to a preferential use of the IM fraction as a substrate, the subcellular distribution of IRS-1, IRS-2, and p85 proteins was analyzed before and after insulin stimulation (Fig. 2 A). For this analysis, equal amounts of protein were loaded in each lane of the SDS-PAGE gel, although the protein distribution in the subcellular fractions was 60, 25, and 15% of the total protein recovered for cytosol, IM, and PM, respectively. When measured at equal protein amounts and in the absence of insulin stimulation, the relative concentration of IRS-1 was approximately 2-fold higher in the IM than in cytosol. Corrected for total protein distribution, this indicates that about 50% of the total IRS-1 is in the IM and about 50% in the cytosol. IRS-2 was also recovered in both IM and cytosolic fractions, but in contrast to IRS-1, the concentration of IRS-2 was 2-fold higher in the cytosol, indicating that 75–80% of this protein is cytosolic. Almost no IRS-1 or IRS-2 was recovered in the PM. Following insulin stimulation, the content of IRS-1 and IRS-2 in the IM declined rapidly with concomitant increases in the cytosol, consistent with previous reports (12Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Ricort J.M. Tanti J.F. Van Obberghen E. Le Marchand Brustel Y. Eur. J. Biochem. 1996; 239: 17-22Crossref PubMed Scopus (85) Google Scholar). The p85 subunit of PI 3-kinase was also recovered in both IM and cytosol and was predominantly cytosolic in the basal state. Following insulin stimulation, there was a 2.5–3-fold increase in p85 in the IM and a concomitant decrease of 50% in cytosol by 1 min, with a gradual return toward the basal state (Fig. 2 A, right panel). The amount of p85 recruited from cytosol to IM represents about 40% of the total p85. p85 docking to IRS-1 and IRS-2 in the IM and cytosol paralleled the phosphorylation of these proteins (see Figs. 1, C andD, and 2 A). Essentially no p85 was recovered in the PM. The subcellular distribution of PI 3-kinase activity associated with IRS-1, IRS-2, and p85 is shown in Fig. 2 B. IRS-1-associated activity increased by 10–20-fold 1 min after insulin stimulation in both the IM fraction and cytosol, with the greatest increase observed in the IM. This reflects the distribution and tyrosine phosphorylation of IRS-1 protein (Figs. 1 C and 2 B). IRS-2-associated activity also increased after insulin stimulation by 5–15-fold. Interestingly, the greatest increase was again predominantly in the IM. This is consistent with the fact that, despite the higher concentration of IRS-2 in the cytosol, the highest level of phosphorylation of IRS-2 occurs in the IM. p85-associated PI 3-kinase activity increased in the IM after insulin stimulation in parallel with the change in p85 protein but did not change in cytosol despite the decrease of p85 protein. A small but detectable amount of p85-associated PI 3-kinase activity was also recovered in the PM fraction, although very little p85 protein was recovered in this compartment. Approximately 75% of the total p85 protein was immunoprecipitated with anti-p85. Interestingly, the same antibody immunoprecipitated approximately 90% of the total pp185 band (i.e. tyrosine-phosphorylated IRS-1/2); however, this accounted for only 13 and 3% of the total IRS-1 and IRS-2, respectively (data not shown). These data indicate that less than 15% of total IRS-1 was tyrosine-phosphorylated 1 min after insulin treatment, and this accounts for the majority of the high molecular weight tyrosine-phosphorylated substrates and most of the activation of PI 3-kinase in 3T3-L1 cells. The PI 3-kinase activity associated with IRS-1 and IRS-2 demonstrated somewhat different patterns than the tyrosine phosphorylation of these proteins (Fig. 3). IRS-1-associated PI 3-kinase in the IM was maximally stimulated by insulin at 1 min after stimulation and remained high in this fraction for the 10 min of study, perhaps due to the high stoichiometry of IRS-1 tyrosine phosphorylation in IM. Cytosolic PI 3-kinase activity associated with IRS-1 increased by more than 10-fold at 1 min and continued to rise gradually. This effect may be due to the translocation of some tyrosine-phosphorylated IRS-1 from IM to cytosol. By comparison, PI 3-kinase associated with IRS-2 was maximal at 1 min after insulin stimulation in both IM and cytosol, and then declined rapidly with time in both the fractions. In addition to activation of PI 3-kinase, IRS-1 and IRS-2 are potentially involved in the Ras signaling pathway by recruiting Grb2. Grb2 was detected in all cellular fractions, with the highest relative concentrations in the IM and cytosol in the unstimulated state (Fig. 4 A). Insulin-induced changes in the subcellular distribution of Grb2 with a 2-fold increase in both PM and IM and a 2-fold decrease in cytosol at 1 min. The changes in Grb2 in the PM and cytosol remained constant over 10 min, whereas the content of Grb2 in IM declined after 1 min of insulin stimulation, a pattern similar to that observed for p85. All three forms of Shc (46, 52, and 66 kDa) could be detected in 3T3-L1 adipocytes, with the 52-kDa form being predominant (Fig.4 B). Under basal conditions, the highest concentration of Shc was in the cytosol, followed by IM and PM. Upon insulin treatment, the 52-kDa isoform of Shc was recruited to the PM with a 3-fold increase by 4 min of stimulation. This was associated with a slight decline in the Shc content in cytosol. Surprisingly, immunoreactive mSOS was recovered almost exclusively in IM, where the concentration of Grb2 is highest, rather than in the PM, where the concentration of Ras is highest (Fig. 4 C). Neither the subcellular distribution of mSOS nor the distribution of Ras was changed by insulin stimulation. Likewise, Akt, one of the serine kinases downstream of PI 3-kinase (28Antonetti D.A. Algenstaedt P. Kahn C.R. Mol. Cell Biol. 1996; 16: 2195-2203Crossref PubMed Scopus (126) Google Scholar,29Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar), was recovered exclusively in cytosol, and its distribution was not changed by insulin stimulation (Fig. 4 C). Gab-1, which has PH and PTB domains and serves as a potential substrate of the insulin receptor (4Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (601) Google Scholar), was also recovered exclusively in cytosol (Fig.4 C). Following insulin stimulation, the mobility of Gab1 appeared to decrease, and there was a dramatic reduction in the signal detected in immunoblots. The exact cause of this decrease is unclear, but it could be related to a change in immunoreactivity, a rapid degradation, or a sequestration in an insoluble compartment, such as the cytoskeleton. From the preceding experiments, it was clear that insulin induced a rapid translocation of IRS-1 from IM to cytosol. This raised the question as to whether there was an insulin-stimulated change in IRS-1 or a change in the IM environment that induces a dissociation of IRS-1. To address this question, we developed an in vitro IRS-1 reassociation assay. IM from control or insulin-treated cells was prepared in the absence or presence of phosphatase inhibitors. The IM was then mixed with cytosol from control or insulin-treated cells prepared in the absence or presence of phosphatase inhibitors. After incubation for 30 min at 30 °C, the IM was reisolated by centrifugation, and the associated IRS-1 and the tyrosine-phosphorylated proteins were analyzed by immunoblotting. Fig. 5 A shows the reassociation assay. First, note that by immunoblotting with anti-IRS-1 and anti-phosphotyrosine, three species of IRS-1 were detected, depending upon the source of the IM (either basal or insulin-stimulated) and whether the cytosol was prepared in the presence (ICy+) or absence (ICy−) of phosphatase inhibitors. These forms of IRS-1 demonstrated progressively reduced mobility on SDS-PAGE and are indicated as IRS-1, IRS-1*, and IRS-1**, respectively. The heterogeneity of migration of IRS-1 results from increasing levels of tyrosine and/or serine/threonine phosphorylation. In the insulin-stimulated cytosol (Fig. 5 A, lane 7, ICy+), the predominant form was IRS-1*, whereas in the basal IM, the predominant form was IRS-1, although some IRS-1* and IRS-1** could be detected (lane 1). As we have previously shown, insulin induces dissociation of IRS-1 from IM; thus, there was a reduced amount of IRS-1 protein in IM from insulin-stimulated cells. However, the predominant form of the IRS-1 protein was the slowest migrating,i.e. IRS-1**, and therefore presumably the most heavily phosphorylated (Fig. 5 A, lane 4). Following incubation of basal IM with insulin-stimulated cytosol (prepared in the presence of phosphatase inhibitors; Fig. 5,ICy+), there was an association of IRS-1* and IRS-1** from the cytosolic fraction with the basal IM (Fig.5 A, lane 2). The incubation of insulin-stimulated IM with insulin-stimulated cytosol resulted in a lower level of IM-associated IRS-1 protein (Fig. 5 A, lane 5). In contrast, when this experiment was performed with insulin-stimulated IM prepared in the absence of phosphatase inhibitors, the IRS-1 present in the cytosol associated with the IM (Fig. 5 B). These data suggest that an insulin-regulated event in the IM is, at least in part, responsible for the insulin-induced dissociation of IRS-1 from the IM. Furthermore, this insulin-stimulated event is most likely a phosphorylation of an IM protein(s), rather than phosphorylation of IRS-1. Consistent with this hypothesis, we found that when IM was prepared in the absence of phosphatase inhibitors, no significant changes in IRS-1 content in IM were observed (Fig.5 B, lanes 1 and 4). Furthermore, the IMs prepared from insulin-treated cells were able to associate with IRS-1* and IRS-1** from insulin-stimulated cytosol prepared in the presence of phosphatase inhibitors (Fig.5 B, lanes 3 and 6). These data support the notion that a phosphorylation event in the IM is important for IRS-1 dissociation. To address the question of whether tyrosine or serine/threonine phosphorylation in IM was responsible for the dissociation event, IM was prepared in the absence or presence of vanadate or okadaic acid or both. As shown in Fig. 6 A, the insulin-induced dissociation of IRS-1 from the IM was observed in membranes that were prepared in the presence of okadaic acid with or without vanadate; however, this effect was not fully preserved in the presence of vanadate without okadaic acid. These observations are consistent with a serine/threonine phosphorylation event in IM responsible for the dissociation of IRS-1. By comparison, the insulin-induced decrease of IRS-2 in IM was observed even if the membranes were prepared without okadaic acid or vanadate, indicating that the lower level of association of IRS-2 with IM may depend on a mechanism different from phosphorylation. To further confirm the serine/threonine phosphorylation dependence of IRS-1 dissociation, IM was first treated with protein phosphatase 2A (PP2A) in the presence or absence of okadaic acid and/or vanadate and then incubated with insulin-stimulated cytosol (Fig. 6 B). Although the incubation in the absence of phosphatase inhibitors alone allowed some IRS-1 to associate with IM, treatment with PP2A markedly restored the association of IRS-1 with the IM. This effect of PP2A was prevented by okadaic acid but not by vanadate. These data are consistent with a serine/threonine phosphorylation event in the IM that modulates the IRS-1/IM interaction. Following insulin binding to its receptor, the insulin receptor tyrosine kinase is rapidly activated and phosphorylates substrate proteins, including IRS-1 and IRS-2 (1Kahn C.R. Diabetes. 1994; 43: 1066-1084Crossref PubMed Scopus (0) Google Scholar). In the present study, we have analyzed the compartmentalization of these two major insulin receptor substrates following insulin stimulation of 3T3-L1 adipocytes and characterized the early signaling to PI 3-kinase. It has been previously noted by several investigators (10Kublaoui B. Lee J. Pilch P.F. J. Biol. Chem. 1995; 270: 59-65Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 12Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Ricort J.M. Tanti J.F. Van Obberghen E. Le Marchand Brustel Y. Eur. J. Biochem. 1996; 239: 17-22Crossref PubMed Scopus (85) Google Scholar, 16Nave B.T. Haigh R.J. Hayward A.C. Siddle K. Shepherd P.R. Biochem. J. 1996; 318: 55-60Crossref PubMed Scopus (129) Google Scholar) and confirmed by the present study that IRS-1 is tyrosine-phosphorylated preferentially in the IM compartment and that there is a redistribution to the cytosol following insulin treatment. This suggests that the subcellular localization of signals might be important for the diversity and specificity in signal transduction. Indeed, the fact that activation of PI 3-kinase occurs at the plasma membrane following stimulation of cells by PDGF as opposed to occurring in the intracellular membranes following insulin stimulation has lead to the hypothesis that subcellular localization of PI 3-kinase may explain the different consequences of PDGF and insulin stimulation of PI 3-kinase on glucose transport (13Ricort J.M. Tanti J.F. Van Obberghen E. Le Marchand Brustel Y. Eur. J. Biochem. 1996; 239: 17-22Crossref PubMed Scopus (85) Google Scholar, 16Nave B.T. Haigh R.J. Hayward A.C. Siddle K. Shepherd P.R. Biochem. J. 1996; 318: 55-60Crossref PubMed Scopus (129) Google Scholar). In the current work, we find that tyrosine phosphorylation of not only IRS-1 but also IRS-2 occurs mainly in the intracellular membrane compartment with relatively low levels in cytosol, implying a localization of signaling by both of these substrates of the insulin receptor. The preferential tyrosine phosphorylation of IRS-1 is explained in part because IRS-1 is located preferentially in the intracellular membranes. However, this cannot explain the increased phosphorylation of IRS-2 in IM, because IRS-2 is preferentially localized in cytosol. Indeed, the total amount of IRS-2 associated with the intracellular membrane is <25% of the total cellular pool, but this fraction accounts for >85% of the tyrosine-phosphorylated IRS-2. This differential localization of IRS-1 and IRS-2 may explain, in part, why IRS-2 contributes less to tyrosine phosphorylation of IRS proteins and p85 association than IRS-1 in 3T3-L1 cells. Because insulin receptors are never recovered in the same membrane fraction as the majority of phosphorylated IRS-1 and IRS-2, an interesting question is how these substrates become phosphorylated. Although it is possible that the IRS proteins become phosphorylated in cytosol and then bind to IM, no such translocation was observed. This suggests that another mechanism must exist by which insulin receptors preferentially phosphorylate IRS-1 and IRS-2 already present in the IM compartment. One possible explanation is that some fraction of the IM vesicles are located in close proximity to the plasma membrane. This would permit the important N-terminal regions of IRS-1 and IRS-2 containing the PH and PTB domains access to the juxtamembrane portion of the insulin receptor and facilitate subsequent phosphorylation (17Myers Jr., M.G. Grammer T.C. Brooks J. Glasheen E.M. Wang L.-M. Sun X.J. Blenis J. Pierce J.H. White M.F. J. Biol. Chem. 1995; 270: 11715-11718Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 18Voliovitch H. Schindler D.G. Hadari Y.R. Taylor S.I. Accili D. Zick Y. J. Biol. Chem. 1995; 270: 18083-18087Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 19Yenush L. Makati K.J. Smith-Hall J. Ishibashi O. Myers Jr., M.G. White M.F. J. Biol. Chem. 1996; 271: 24300-24306Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 20Eck M.J. Dhe-Paganon S. Trub T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Despite this possibility, only about 15% of IRS-1 and even less IRS-2 was tyrosine-phosphorylated by 1 min after insulin stimulation (the time of maximal tyrosine phosphorylation), perhaps reflecting the large cytosolic pools of these proteins. The ability of the p85 subunit of PI 3-kinase to bind to IRS protein generally parallels the level of tyrosine phosphorylation in subcellular fractions. Thus, p85 association with IRS-1 is greater than that with IRS-2. Also, p85 association to both IRS-1 and IRS-2 is greater in the IM than in cytosol. This results in an insulin-stimulated recruitment of p85 from cytosol to the IM. The return of p85 from the IM to the cytosol several minutes post-insulin stimulation may be the result of the translocation of IRS-1 and IRS-2 from the IM to cytosol and the concomitant decrease in tyrosine phosphorylation of these substrates. Because the level of tyrosine phosphorylation of IRS-1 in the IM is relatively stable over the first 10 min following stimulation with insulin (despite a progressive decrease in IRS-1 protein), we can conclude that the apparent stoichiometry of IRS-1 tyrosine phosphorylation in the IM must be increasing, whereas that in cytosol is decreasing. This suggests that the activated insulin receptors continue to phosphorylate IRS-1 in the IM, leading to a continuous recruitment of p85 to the IM. Although the tyrosine-phosphorylated IRS-1 translocated to cytosol undergoes a relatively rapid dephosphorylation, the PI 3-kinase associated with IRS-1 in cytosol remains relatively stable over 10 min. This suggests that the dephosphorylation of IRS-1 in cytosol may preferentially occur at sites other than PI 3-kinase binding sites (data not shown). By comparison, the phosphorylation of IRS-2 and its docking to PI 3-kinase decrease rapidly both in the IM and cytosol following insulin stimulation. This is consistent with the recent report that tyrosine phosphorylation and PI 3-kinase association of IRS-2 are more transient than those of IRS-1 in L6 cells (30Ogihara T. Shin B.-C. Anai M. Katagiri H. Inukai K. Funaki M. Fukushima Y. Ishihara H. Takata K. Kikuchi M. Yazaki Y. Oka Y. Asano T. J. Biol. Chem. 1997; 272: 12868-12870Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The ability of IRS-1 to associate with the IM and have a higher stoichiometry of tyrosine-phosphorylation and the possible relative resistance to dephosphorylation of IRS-1 at the PI 3-kinase binding sites result in a greater contribution of IRS-1 to insulin-stimulated PI 3-kinase in both the IM and cytosol. A working model of the dynamics of IRS protein movement in response to insulin is depicted in Fig. 7. The most important aspect of the current study is the ability to reconstitute the association of IRS-1 to IM in vitro. Results from these experiments suggest that the translocation of IRS-1 from the IM to cytosol is due to a decreased ability of the IM to associate with IRS-1 and is regulated by an insulin-induced serine/threonine phosphorylation of some component in the IM. Thus, the IM from insulin-stimulated cells has a decreased ability to associate with IRS-1. This is true whether the IRS-1 is obtained from basal or insulin-stimulated cells and whether or not the IRS-1 is tyrosine- or serine/threonine-phosphorylated (as judged by both immunoblotting and gel mobility). We also noted that exclusion of okadaic acid from the homogenization buffer (which allows dephosphorylation of serine/threonine residues in the IM) prevents the dissociation of IRS-1. Likewise, treatment of the insulin-stimulated IM with PP2A restores its ability to associate with IRS-1. It has been suggested that the association between membranes and IRS-1 most likely involves the PH and PTB domains of IRS-1, because these domains are capable of many protein-protein and protein-lipid interactions (29Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). Addition of a recombinant IRS-1 fragment containing only the PH and PTB domains in the reconstitution assay had no effect on IRS-1 interaction with the IM (data not shown). Although this does not rule out a role for the PH/PTB region in interacting with IM, it does suggest that the recombinant protein alone is insufficient to compete with endogenous IRS-1 for association with IM. Further studies will be needed to define the region of IRS-1 responsible for association with the IM and to detect the possible IRS-1 binding component(s) present in the IM. The dissociation of IRS-2 from IM appears to be modulated differently because it occurs even in the absence of phosphatase inhibitors. We also examined the location of Grb2, Shc, mSOS, and Ras as components of the Ras-mitogen-activated protein kinase pathway and Akt as a downstream signaling partner of PI 3-kinase (31Franke T.F. Yang S. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1827) Google Scholar, 32Bergering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1880) Google Scholar). Gab-1, which has a PH domain (4Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (601) Google Scholar) and has been reported to be an insulin receptor substrate in some cells, is exclusively in the cytosol of 3T3-L1 cells. Furthermore, Gab 1 is not tyrosine-phosphorylated, nor does it change subcellular location upon insulin stimulation. Insulin-stimulated tyrosine phosphorylation of Shc was observed exclusively in the plasma membrane, and this was associated with a minor translocation of the 52-kDa isoform of Shc from the cytosol to the plasma membrane. Insulin-stimulated tyrosine-phosphorylation of Shc exclusively in the plasma membrane was also observed using freshly isolated rat adipocytes (data not shown). In contrast, Grb2 is recruited from cytosol primarily to the IM with a lesser degree of association with the plasma membrane. The recruitment of Grb2 to the IM reaches a maximum 1 min after insulin stimulation and then decreases in a pattern similar to that of p85. In addition, whereas Ras is exclusively in the plasma membrane, mSOS is, surprisingly, exclusively in the IM and does not change after insulin stimulation. Likewise, immunoreactive Akt is exclusively in cytosol and does not change after insulin stimulation. Although, several mechanisms for activation of Akt have been proposed, including phosphorylation and binding to phosphatidylinositol 3,4-bisphosphate through the PH domain of Akt (33Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2517) Google Scholar, 34Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1305) Google Scholar, 35Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell Biol. 1997; 17: 338-344Crossref PubMed Scopus (447) Google Scholar), membrane localization would be predicted to be important because attachment of the src myristoylation signal to target Akt to the membrane constitutively activates Akt (36Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). The failure to find Akt in membrane fractions may reflect a low affinity interaction and dissociation during fractionation or pools with different antibody reactivity. In summary, molecules involved in insulin signaling, such as IRS-1, IRS-2, p85, Grb2, and Shc, exhibit distinctive compartmentalization and undergo rapid changes in the subcellular localization in 3T3-L1 adipocytes following insulin stimulation. The compartmentalization and redistribution of these molecules may help explain how shared signaling components can elicit specific and unique downstream events for various growth factors. It may also explain how a single hormone, such as insulin, may elicit effects in one cell that differ from those produced by the same hormone in another cell type. We thank Terri-Lyn Bellman and Natalie Zahr for excellent secretarial assistance." @default.
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• W2002398354 title "Dynamics of Insulin Signaling in 3T3-L1 Adipocytes" @default.
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