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14-3-3β Protein Associates with Insulin Receptor Substrate 1 and Decreases Insulin-stimulated Phosphatidylinositol 3′-Kinase Activity in 3T3L1 Adipocytes*

Open AccessPublished:January 09, 1998DOI:https://doi.org/10.1074/jbc.273.2.940
      The 14-3-3 protein family has been implicated in growth factor signaling. We investigated whether 14-3-3 protein is involved in insulin signaling in 3T3L1 adipocytes. A significant amount of insulin receptor substrate 1 (IRS-1) was immunodetected in the immunoprecipitate with anti-14-3-3β antibody at the basal condition. 100 nm insulin increased the amount of IRS-1 in the immunoprecipitate 2.5-fold. The effect of insulin was abolished by 100 nm wortmannin. An in vitro binding study revealed that glutathione S-transferase-14-3-3β fusion protein directly associates with recombinant IRS-1. Pretreatment of recombinant IRS-1 with alkaline phosphatase clearly decreased this association. Because the recombinant IRS-1 was not phosphorylated on its tyrosine residues, the results suggest that serine/threonine phosphorylation of IRS-1 is responsible for the association. When the cells are treated with insulin, phosphatidylinositol 3′-kinase (PI3K) is supposed to complex either 14-3-3β-IRS-1 or IRS-1. The 14-3-3β-IRS-1-PI3K and IRS-1-PI3K complexes were separately prepared by a sequential immunoprecipitation, first with anti-14-3-3β and then with anti-IRS-1 antibodies. The specific activity of the PI3K in the former was approximately half of that in the latter, suggesting that 14-3-3β protein bound to IRS-1 inhibits insulin-stimulated lipid kinase activity of PI3K in 3T3L1 adipocytes.
      Insulin promotes the rapid autophosphorylation of its receptor β-subunits and tyrosine phosphorylation of several cytoplasmic proteins such as Shc, pp60, Gab-1, and insulin receptor substrate (IRS)
      The abbreviations used are: IRS, insulin receptor substrate; PI3K, phosphatidylinositol 3′-kinase; GST, glutathione S-transferase; IP, immunoprecipitation; difluoroPmp, difluorophosphonometyl phenylalanine; Fmoc,N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; TBS, Tris-buffered saline.
      1The abbreviations used are: IRS, insulin receptor substrate; PI3K, phosphatidylinositol 3′-kinase; GST, glutathione S-transferase; IP, immunoprecipitation; difluoroPmp, difluorophosphonometyl phenylalanine; Fmoc,N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; TBS, Tris-buffered saline.
      1 and 2. IRS-1 plays a central role in insulin signaling. The protein contains a pleckstrin homology domain, a phosphotyrosine-binding domain that binds to NPXY motif of the insulin receptor β-subunit and multiple tyrosine residues that are potential phosphorylation sites. The tyrosine-phosphorylated IRS-1 associates with several Src homology 2 domain-containing proteins including Grb-2, SHP-2, Nck, Fyn, and the 85-kDa subunit (p85) of phosphatidylinositol 3′-kinase (PI3K). Associations of these molecules with IRS-1 are believed to further activate downstream signaling systems, including mitogen-activated protein kinase and PI3K cascades, that promote mitogenic and metabolic effects of insulin (for review see Refs.
      • Cheatham B.
      • Kahn C.R.
      and
      • Myers Jr., M.G.
      • White M.F.
      ).
      14-3-3 protein family, first discovered as acidic proteins in the brain, is highly conserved in animals and plants. At least nine mammalian isoforms have been identified, and six are expressed ubiquitously (
      • Watanabe M.
      • Isobe T.
      • Ichimura T.
      • Kuwano R.
      • Takahashi Y.
      • Kondo H.
      • Inoue Y.
      ). Originally, 14-3-3 proteins have been shown to be functional as regulators of tryptophan and tyrosine hydroxylases as well as protein kinase C (for review see Ref.
      • Aitken A.
      ). However, recently 14-3-3 proteins were found to associate oncogene products, including Raf-1 (
      • Fantl W.J.
      • Muslin A.J.
      • Kikuchi A.
      • Martin J.A.
      • MacNicol A.M.
      • Gross R.W.
      • Williams L.T.
      ,
      • Fu H.
      • Xia K.
      • Pallas D.C.
      • Cui C.
      • Conroy K.
      • Narsimhan R.P.
      • Mamon H.
      • Collier R.J.
      • Roberts T.M.
      ,
      • Freed E.
      • Symons M.
      • Macdonald S.G.
      • McCormick F.
      • Ruggieri R.
      ,
      • Irie K.
      • Gotoh Y.
      • Yashar B.M.
      • Errede B.
      • Nishida E.
      • Matsumoto K.
      ,
      • Yamamori B.
      • Kuroda S.
      • Shimizu K.
      • Fukui K.
      • Ohtsuka T.
      • Takai Y.
      ,
      • Li S.
      • Janosch P.
      • Tanji M.
      • Rosenfeld G.C.
      • Waymire J.C.
      • Mischak H.
      • Kolch W.
      • Sedivy J.M.
      ,
      • Michaud N.R.
      • Fabian J.R.
      • Mathes K.D.
      • Morrison D.K.
      ), Bcr-Abl, Bcr (
      • Reuther G.W.
      • Fu H.
      • Cripe L.D.
      • Collier R.J.
      • Pendergast A.M.
      ,
      • Braselmann S.
      • McCormick F.
      ), polyoma middle T antigen (
      • Pallas D.C.
      • Fu H.
      • Haehnel L.C.
      • Weller W.
      • Collier R.J.
      • Roberts T.M.
      ), and cell cycle control proteins such as Cdc25 phosphatases (
      • Conklin D.S.
      • Galaktionov K.
      • Beach D.
      ). The interaction of Raf with 14-3-3 leads to Raf activation in severalin vivo systems (
      • Fantl W.J.
      • Muslin A.J.
      • Kikuchi A.
      • Martin J.A.
      • MacNicol A.M.
      • Gross R.W.
      • Williams L.T.
      ,
      • Freed E.
      • Symons M.
      • Macdonald S.G.
      • McCormick F.
      • Ruggieri R.
      ,
      • Irie K.
      • Gotoh Y.
      • Yashar B.M.
      • Errede B.
      • Nishida E.
      • Matsumoto K.
      ). Moreover, the importance of 14-3-3 proteins in signal transduction is suggested by the reports that 14-3-3 protein interacts with PI3K (
      • Bonnefoy-Berard N.
      • Liu Y.
      • von Willebrand M.
      • Sung A.
      • Elly C.
      • Mustelin T.
      • Yoshida H.
      • Ishizaka K.
      • Altman A.
      ) and glycoprotein Ib-IX (
      • Du X.
      • Harris S.J.
      • Tetaz T.J.
      • Ginsberg M.H.
      • Berndt M.C.
      ). However, both the mechanism and the physiological role of protein-protein interactions mediated by 14-3-3 proteins are still unclear.
      The aim of the present study is to investigate whether the 14-3-3 protein is involved in the insulin signaling pathway. We showed that 14-3-3β protein directly binds to IRS-1 and that insulin increases this binding via a wortmannin-sensitive pathway in 3T3L1 adipocytes. We also demonstrated that the 14-3-3β protein negatively regulates insulin-stimulated activity of PI3K.

      RESULTS AND DISCUSSION

      To investigate whether 14-3-3β protein is involved in the insulin signaling pathway, total cell lysates of 3T3L1 adipocytes were immunoprecipitated with anti-14-3-3β antibody, and the immune complexes were subjected to immunoblots with antibodies against molecules implicated in insulin signaling. We found that a significant amount of IRS-1 was immunodetected in the immunoprecipitate at the basal condition. 100 nm insulin increased 2.5-fold (basalversus insulin-stimulated condition; 5.30 ± 0.41versus 13.57 ± 1.16 arbitrary units, n = 6,p < 0.01) the amount of IRS-1 in the immunoprecipitate (Fig. 1 A, lanes 1and 2). The increase was detected very rapidly after insulin treatment, reaching to the plateau at 1 min (data not shown). The effect of insulin was abolished when the cells were pretreated with 100 nm wortmannin (Fig. 1 A, lane 3). However, wortmannin did not affect the amount of immunoprecipitated IRS-1 at the basal condition (data not shown). To confirm these results, the total cell lysates were immunoprecipitated with anti-IRS-1 antibody, and the immune complexes were subjected to immunoblotting with anti-14-3-3β antibody (Fig. 1 B). Consistent with the former results, 14-3-3β proteins were immunodetected in the immunoprecipitate at the basal condition. Insulin increased the amount of immunoprecipitated 14-3-3β proteins. The effect of insulin was reduced by wortmannin pretreatment.
      Figure thumbnail gr1
      Figure 1Insulin-induced association of 14-3-3β protein with IRS-1 in 3T3L1 adipocytes. 3T3L1 adipocytes were pretreated with (lanes 3 and 8) or without (lanes 1, 2, 4–7, and 9) 100 nm wortmannin for 20 min at 37 °C. The cells were then treated with (lanes 2, 3, 7, and8) or without (lanes 1, 4–6, and9) 100 nm insulin for 3 min at 37 °C. 2 mg of the total cell lysates were immunoprecipitated with anti-14-3-3β antibody (lanes 1–3 and 9), anti-IRS-1 antibody (lanes 5–8), or control IgG (lane 4). The immunoprecipitates were subjected to immunoblots with anti-IRS-1 (A) or anti-14-3-3β (B) antibody. Each representative photograph is shown. Blot, immunoblot; α-IRS-1, anti-IRS-1 antibody; α-14-3-3β, anti-14-3-3β antibody;CO, control IgG.
      To ensure that 14-3-3β protein associates with IRS-1, in vitro association experiments were performed using a GST-14-3-3β fusion protein. GST-14-3-3β protein was incubated with total cell lysates of 3T3L1 adipocytes treated with or without insulin, followed by glutathione-Sepharose 4B beads precipitation. The precipitates were subjected to immunoblotting using anti-IRS-1 antibody (Fig.2 A). The GST-14-3-3β protein (but not GST alone) associated with endogenous IRS-1 in the cell lysates at the basal condition. Insulin increased the association by 2-fold. These results are in agreement with the data obtained fromin vivo studies (Fig. 1).
      Figure thumbnail gr2
      Figure 2In vitro association of 14-3-3β protein with IRS-1. A, 3T3L1 adipocytes were pretreated with (lanes 2 and 4) or without (lanes 1and 3) 100 nm insulin for 3 min at 37 °C. 2 mg of the total cell lysates were incubated with 50 pmol of GST alone (lanes 1 and 2) or GST-14-3-3β fusion protein (lanes 3 and 4) for 1 h, followed by the precipitation with glutathione-Sepharose 4B beads for another hour at 4 °C. The precipitates were washed and subjected to an immunoblot with anti-IRS-1 antibody. B, 400 ng of rat recombinant IRS-1 protein were incubated with 50 pmol of either GST alone (lane 6) or GST-14-3-3β fusion protein (lane 7) in lysis buffer for 1 h and then with glutathione-Sepharose 4B beads for another hour at 4 °C. The precipitates were washed and subjected to an immunoblot with anti-IRS-1 antibody. One-quarter the amount of input recombinant IRS-1 was subjected to the immunoblot as a positive control (lane 5). C, 15 units of either active (lanes 9 and 11) or heat-inactivated (lanes 8 and 10) calf intestine alkaline phosphatase were incubated with 400 ng of rat recombinant IRS-1 protein for 30 min at 25 °C and then heat-inactivated for 10 min at 70 °C. The recombinant IRS-1 proteins were then incubated with 50 pmol of either GST alone (lanes 10 and 11) or GST-14-3-3β fusion protein (lanes 8 and 9) in lysis buffer for 1 h and then with glutathione-Sepharose 4B beads for another hour at 4 °C. The precipitates were washed and subjected to an immunoblot with anti-IRS-1 antibody. The results show one representative out of three independent experiments. GS4B, glutathione-Sepharose 4B beads; rIRS-1, recombinant IRS-1;ALP, calf intestine alkaline phosphatase; prep., precipitation.
      To further address whether the direct association of 14-3-3β protein with IRS-1 can occur, in vitro reconstitution experiments were performed using recombinant IRS-1 and GST-14-3-3β fusion protein. We found that the GST-14-3-3β protein directly associates with recombinant IRS-1 (Fig. 2 B). Recently, it has been determined that 14-3-3 protein binds to phosphoserine (pS)-containing motifs by the peptide binding and peptide mapping analysis (
      • Muslin A.J.
      • Tanner J.W.
      • Allen P.M.
      • Shaw A.S.
      ,
      • Zha J.
      • Harada H.
      • Yang E.
      • Jockel J.
      • Korsmeyer S.J.
      ). Therefore, to test whether phophorylated residues of recombinant IRS-1 are involved in the association with 14-3-3β protein, effects of alkaline phosphatase on the association were determined. Pretreatment of the recombinant IRS-1 with alkaline phosphatase clearly decreased this association (Fig. 2 C). The results indicate that 14-3-3β protein directly binds to IRS-1 via its phosphorylated residues, probably phosphorylated serine/threonine residues, because the recombinant IRS-1 that was used for the experiment was not phosphorylated on tyrosine residues. These in vitroreconstitution experiments could explain the association of 14-3-3β protein with IRS-1 at the basal condition. However, mechanisms of the insulin-induced increase in the association of 14-3-3β protein with IRS-1 remains unknown. It was shown that RSXpSXP, RXRXXpS and a cluster of serine residues in the α-helix are the binding motifs for 14-3-3 proteins (
      • Muslin A.J.
      • Tanner J.W.
      • Allen P.M.
      • Shaw A.S.
      ,
      • Zha J.
      • Harada H.
      • Yang E.
      • Jockel J.
      • Korsmeyer S.J.
      ,
      • Du X.
      • Fox J.E.
      • Pei S.
      ). Interestingly, IRS-1 contains six RXRXXS motifs (but no RSXSXP motifs) in its amino acid sequence. Four of them are in the phosphotyrosine-binding domain, and the others are located in the region containing the YXXM motifs. Moreover, it was reported that IRS-1 is a substrate for serine kinase of PI3K activated by insulin (
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ). Taken together with our results, it is tempting to speculate that IRS-1 binds to 14-3-3β protein via its phosphorylated serine residues such as RXRXXpS motif, and insulin treatment results in the activation of serine kinase of PI3K, which in turn further phosphorylates the serine residues of IRS-1, leading to more association with 14-3-3β protein. This could be a reason why wortmannin inhibits insulin-induced increase of the IRS-1–14-3-3β complex (Fig. 1). Of course, other molecules downstream from PI3K, for example, protein kinase B, which is another serine/threonine kinase, may be involved in the insulin-induced increase of the IRS-1–14-3-3β complex (
      • Burgering B.M.T.
      • Coffer P.J.
      ). Further study will be necessary to clarify this point.
      An alternative possibility is that insulin-stimulated association of 14-3-3β proteins with IRS-1 is mediated by PI3K. It is well known that tyrosine-phosphorylated IRS-1 upon insulin stimulation associates directly with the p85 subunit of PI3K through pYMXM and pYXXM motifs, resulting in the activation of PI3K (
      • Myers Jr., M.G.
      • White M.F.
      ). It has been reported that insulin induces serine-phosphorylation of p85 by PI3K itself (
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ,
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry J.M.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ). As expected, p85 was detected in the immunoprecipitates with anti-14-3-3β antibody mainly upon insulin treatment and little in the basal condition. This band was decreased in the presence of wortmannin (Fig.3 A). If 14-3-3β protein associates with PI3K directly, insulin apparently increases the amount of IRS-1 immunoprecipitated with 14-3-3β antibody via PI3K. Therefore, to explore this possibility, we activated PI3K in vitro without the insulin receptor-IRS-1 system. pYMXM- or difluoroPmpMXM-containing peptide was incubated with the cytosolic fraction of untreated 3T3L1 adipocytes, and the mixture was immunoprecipitated with anti-p85 antibody (
      • Otaka A.
      • Miyoshi K.
      • Kaneko M.
      • Tamamura H.
      • Fujii N.
      • Nomizu M.
      • Burke Jr., T.R.
      • Roller P.P.
      ,
      • Herbst J.J.
      • Andrews G.C.
      • Contillo L.G.
      • Singleton D.H.
      • Genereux P.E.
      • Gibbs E.M.
      • Lienhard G.E.
      ). The immunoabsorbed PI3K was further phosphorylated by adding ATP and manganese and then subjected to in vitro association experiments with GST-14-3-3β protein. Although both pYMXM- and difluoroPmpMXM-containing peptide activated the lipid kinase activity of PI3K (Fig. 3 B) and also increased phosphorylation of p85 (Fig. 3 C), neither pYMXM- nor difluoroPmpMXM-containing peptide increased the association of GST-14-3-3β protein with PI3K in the absence of IRS-1 molecule (Fig. 3 D, upper panel). It is thus unlikely that 14-3-3β protein directly associates with PI3K in 3T3L1 adipocytes, accounting for the insulin-stimulated increase of IRS-1 in the immunoprecipitates with anti-14-3-3β antibody. Recently, it was reported that 14-3-3τ protein directly binds to PI3K in human T lymphocytes (
      • Bonnefoy-Berard N.
      • Liu Y.
      • von Willebrand M.
      • Sung A.
      • Elly C.
      • Mustelin T.
      • Yoshida H.
      • Ishizaka K.
      • Altman A.
      ). The difference from our results may be due to the tissue, 14-3-3 isoform, or species specificity. It is of interest to note that human but not mouse p85 of PI3K contains RSXSXP motif in the breakpoint cluster region homologue domain.
      Figure thumbnail gr3
      Figure 3No association of 14-3-3β protein with PI3K in the absence of IRS-1. A, 3T3L1 adipocytes were pretreated with (lane 3) or without (lanes 1 and2) 100 nm wortmannin for 20 min at 37 °C. The cells were then treated with (lanes 2 and 3) or without (lane 1) 100 nm insulin for 3 min at 37 °C. 2 mg of the total cell lysates were immunoprecipitated with anti-14-3-3β antibody. The immunoprecipitates were subjected to an immunoblot with anti-p85 antibody. The numbers on theright show the positions of 97- and 66-kDa markers.B, 1.5 mg of the cytosol fraction of untreated 3T3L1 adipocytes was incubated with 10 μm of either YMXM- (lane 4), pYMXM- (lane 5), or difluoroPmpMXM-containing (lane 6) peptides for 20 min at 25 °C and immunoprecipitated with anti-p85 antibody. The immunoprecipitated PI3K was subjected to lipid kinase assay, as described under “Experimental Procedures.” C, a part of the immunoprecipitated PI3K in B was incubated with 20 μCi of [γ-32P]ATP in buffer A for 10 min at 25 °C and washed. The samples were subjected to SDS-polyacrylamide gel electrophoresis and autoradiography to visualize the p85 autophosphorylation. D, a part of the immunoprecipitated PI3K in B was further phosphorylated in 50 μl of buffer A for 10 min at 25 °C and then incubated with 30 pmol of GST-14-3-3β proteins in 200 μl of lysis buffer for 1 h at 4 °C. The immune complexes were washed and subjected to immunoblots with anti-GST (upper panel) or anti-p85 (lower panel) antibody. One-third the amount of input GST-14-3-3β protein was shown as a positive control (lane 14), and preimmune IgG was used for a negative control (lane 10). The results show one representative out of three independent experiments.α-14-3-3β, anti-14-3-3β antibody; α-p85, anti-p85PI3K antibody; CO, preimmune IgG;Wort, wortmannin; Y, YMXM-peptide;P, pYMXM-peptide; F, difluoroPmpMXM-peptide; PI3P, phosphatidylinositol 3-phosphate.
      Because less than half the amount of total IRS-1 associates with 14-3-3β protein when the cells are treated with insulin (Fig.1 A), PI3K may complex either 14-3-3β-IRS-1 or IRS-1. To address the physiological significance of the 14-3-3β-IRS-1-PI3K complex, the 14-3-3β-IRS-1-PI3K and IRS-1-PI3K complexes were separately prepared by a sequential immunoprecipitation (Fig.4). The total cell lysates were first immunoprecipitated with anti-14-3-3β antibody (Fig. 4, lanes 1 and 2), and then half of the supernatant was immunoprecipitated with anti-IRS-1 antibody (Fig. 4, lanes 3and 4). The lipid kinase activity upon insulin stimulation in the former (14-3-3β-IRS-1-PI3K complex; Fig. 4 A,lane 2) was about 25% of that in the latter (IRS-1-PI3K complex; Fig. 4 A, lane 4, and TableI). A part of each sample in Fig.4 A was also subjected to immunoblots with anti-IRS-1 (Fig.4 B), anti-phosphotyrosine (Fig. 4 C), anti-p85PI3K (Fig. 4 D), or anti-14-3-3β (Fig.4 E) antibody. After insulin stimulation, the amounts of IRS-1, tyrosine-phosphorylated p185, and p85PI3K in the first immunoprecipitates (Fig. 4, B, C, andD, lanes 2) were about 50% of those in the second immunoprecipitates (Fig. 4, B, C, andD, lanes 4, and Table I). When normalized by the amount of p85, insulin-induced lipid kinase activity was significantly lower in the first immunoprecipitate compared with that in the second immunoprecipitate. Both the tyrosine phosphorylation state of IRS-1 and the association of p85 with IRS-1 were similar in the first and the second immunoprecipitates. Thus 14-3-3β protein appeared to be a negative regulator for PI3K in insulin signaling system. Here, the question is raised as to how 14-3-3β protein regulates PI3K. In preliminary studies using immunoblots with anti-p110αPI3Kantibody, we found that the ratio of p110α catalytic subunit to p85 regulatory subunit of PI3K was decreased in the first immunoprecipitate compared with that in the second immunoprecipitate.
      A. Kosaki, K. Yamada, and H. Kuzuya, unpublished observation.
      Because the antibody against the p110α subunit was low in the affinity, this finding must be further confirmed using different antibodies that work well in the immunoblotting. However, at this point, it should be emphasized that PI3K activity is reduced in the 14-3-3β-IRS-1-PI3K complex when normalized by the amount of p85 subunit.
      Table ILipid kinase activity of PI3K bound to the IRS-1-14-3-3β complex
      Lipid kinase or immunoblotFirst IP anti-14–3-3βSecond IP anti-IRS-1
      %
      Lipid kinase24.9 ± 7.4
      p < 0.02 versus the values of IRS-1, pTyr, and p85 in the first IP.
      100
      IRS-156.6 ± 4.7100
      pTyr50.1 ± 2.8100
      p8551.4 ± 3.1100
      14–3-3β1001.4 ± 0.3
      1-a p < 0.02 versus the values of IRS-1, pTyr, and p85 in the first IP.
      The physiological role of protein-protein interactions mediated by 14-3-3 proteins is still under investigations. Recently, 14-3-3 protein has been shown to be a modulator of kinases including Raf-1, protein kinase C, and PI3K (
      • Aitken A.
      ,
      • Bonnefoy-Berard N.
      • Liu Y.
      • von Willebrand M.
      • Sung A.
      • Elly C.
      • Mustelin T.
      • Yoshida H.
      • Ishizaka K.
      • Altman A.
      ). Furthermore, an importance of 14-3-3 protein as a chaperone as well as an adapter molecule has recently been proposed (
      • Jones D.H.
      • Ley S.
      • Aitken A.
      ). Indeed, it was reported that 14-3-3 protein mediates the interaction of Bcr and Raf and the interaction of A20 protein and c-Raf by functioning as a bridging adapter molecule (
      • Braselmann S.
      • McCormick F.
      ,
      • Vincenz C.
      • Dixit V.M.
      ). Therefore, we tested whether 14-3-3β protein is acting as an adapter molecule in between IRS-1 and Raf-1. Total cell lysates of 3T3L1 adipocytes treated with or without insulin were immunoprecipitated with anti-IRS-1 antibody, and the immune complexes were subjected to immunoblotting with anti-Raf-1 antibody. However, the results did not support the presence of interaction between IRS-1 and Raf-1 via 14-3-3β protein in the 3T3L1 adipocytes, despite the fact that the interaction between 14-3-3β and Raf-1 protein was clearly observed.2 In conclusion, in this study we showed that the 14-3-3 protein is another molecule bound to IRS-1 that modulates PI3K in insulin signaling system in 3T3L1 adipocytes.

      Acknowledgments

      We thank Dr. Masahiko Watanabe for a gift of 14-3-3β cDNA, Dr. Terrence R. Burke, Jr. for a gift of difluorophosphonometyl phenylalanine, and the Radioisotope Research Center of Kyoto University (Kyoto, Japan) for helpful support. We also thank Michiyo Amaoka and Yumi Tanaka for technical assistance and Kuniko Fukami for secretarial assistance.

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