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Inhibition of Phosphatidylinositol 3-Kinase Activity by Adenovirus-mediated Gene Transfer and Its Effect on Insulin Action*

Open AccessPublished:July 17, 1998DOI:https://doi.org/10.1074/jbc.273.29.18528
      Phosphatidylinositol 3-kinase (PI 3-K) is implicated in cellular events including glucose transport, glycogen synthesis, and protein synthesis. It is activated in insulin-stimulated cells by binding of the Src homology 2 (SH2) domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1), and, others. We have previously shown that IRS-1-associated PI 3-kinase activity is not essential for insulin-stimulated glucose transport in 3T3-L1 adipocytes, and that alternate pathways exist in these cells. We now show that adenovirus-mediated overexpression of the p85N-SH2 domain in these cells behaves in a dominant-negative manner, interfering with complex formation between endogenous PI 3-K and its SH2 binding targets. This not only inhibited insulin-stimulated IRS-1-associated PI 3-kinase activity, but also completely blocked anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. This resulted in inhibition of insulin-stimulated glucose transport, glycogen synthase activity and DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules Akt and p70 S6 kinase was inhibited. However, co-expression of a membrane-targeted p110CAAX with the p85N-SH2 protein rescued glucose transport, supporting our argument that the p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase activity, and, that the signaling pathways downstream of PI 3-kinase are intact. Unexpectedly, GTP-bound Ras was elevated in the basal state. Since p85 is known to interact with GTPase-activating protein in 3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out cellular GTPase-activating protein by direct association, such that it is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced mitogen-activated protein kinase phosphorylation was inhibited. Thus, PI 3-kinase may be required for this action at a step independent of and downstream of Ras. We conclude that, in 3T3-L1 adipocytes, non-IRS-1-associated PI 3-kinase activity is crucial for insulin's metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a variety of insulin's ultimate biological effects.
      Insulin binding to its cell surface receptors initiates diverse metabolic and mitogenic signals by activation of a complex signaling cascade of protein tyrosine and serine/threonine kinases, as well as lipid kinases (
      • Cheatham B.
      • Kahn C.R.
      ,
      • Braselmann S.
      • Palmer T.M.
      • Cook S.J.
      ). Phosphatidylinositol (PI)
      The abbreviations used are: PI, phosphatidylinositol; GAP, GTPase-activating protein; GLUT4, insulin-responsive glucose transporter; IRS, insulin receptor substrate; MAP, microtubule-associated protein; MAPK, microtubule-associated protein kinase; p85, 85-kDa regulatory subunit of PI 3-kinase; BrdUrd, bromodeoxyuridine; m.o.i., multiplicity of infection; bp, base pair(s); β-gal, β-galactosidase; 2-DOG, 2-deoxyglucose; SH, Src homology; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; α-MEM, α-minimal essential medium; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; GSK3, glycogen synthase kinase 3.
      1The abbreviations used are: PI, phosphatidylinositol; GAP, GTPase-activating protein; GLUT4, insulin-responsive glucose transporter; IRS, insulin receptor substrate; MAP, microtubule-associated protein; MAPK, microtubule-associated protein kinase; p85, 85-kDa regulatory subunit of PI 3-kinase; BrdUrd, bromodeoxyuridine; m.o.i., multiplicity of infection; bp, base pair(s); β-gal, β-galactosidase; 2-DOG, 2-deoxyglucose; SH, Src homology; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; α-MEM, α-minimal essential medium; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; GSK3, glycogen synthase kinase 3.
      3-kinase (PI 3-kinase), a dual protein and lipid kinase is a heterodimeric enzyme composed of a 110-kDa catalytic subunit (p110) associated with an 85-kDa regulatory subunit (p85). Two isoforms of the catalytic subunit (p110α and p110β) and several isoforms of the regulatory subunit (p55α, p55PIK, p85α, and p85β) have been cloned so far. The regulatory subunit contains several well known functional domains: one Src homology 3 (SH3) domain, homology to the breakpoint cluster region (bcr) gene, two proline-rich motifs, and two Src homology region 2 (SH2) domains (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ). The p85α isoform is insulin-responsive and is predominantly expressed in 3T3-L1 adipocytes (
      • Baltensperger K.
      • Kozma L.M.
      • Jaspers S.R.
      • Czech M.P.
      ).
      The insulin receptor is a tyrosine kinase, which, when activated by insulin, phosphorylates cellular substrates such as IRS-1, IRS-2, IRS-3, IRS-4 and the protein Shc, etc. (
      • Myers Jr., M.G.
      • White M.F.
      ,
      • Waters S.B.
      • Pessin J.E.
      ,
      • Lavan B.E.
      • Lane W.S.
      • Lienhard G.E.
      ,
      • Lavan B.E.
      • Fantin V.R.
      • Chang E.T.
      • Lane W.S.
      • Keller S.R.
      • Lienhard G.E.
      ). IRS-1 is the most well characterized among members of the IRS family. Following insulin stimulation, the phosphorylated YXXM motifs in IRS-1 binds to the SH2 domains of p85 stimulating the lipid kinase activity of the p110 subunit. Through the same SH2 mechanism, PI 3-kinase can associate with other proteins such as IRS-2–4, and the insulin receptor itself (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.-J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Backer J.M.
      • Myers Jr., M.G.
      • Sun X.-J.
      • Chin D.J.
      • Shoelson S.E.
      • Miralpeix M.
      • White M.F.
      ). PI 3-kinase phosphorylates phosphatidylinositol (PI), phosphatidylinositol-4-monophosphate, and phosphatidylinositol-4,5-bisphosphate on the D-3 position of the inositol ring producing phosphatidylinositol 3-monophosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-triphosphate, respectively. Insulin induces the production of D3-phosphorylated phosphoinositides through stimulation of PI 3-kinase activity in various tissues and cultured cells.
      Insulin is a pleiotropic hormone that initiates a variety of biologic effects through a complex set of signaling cascades. As a general view, however, two main branches of the insulin signaling pathway can be activated in most cell types: one is controlled predominantly by PI 3-kinase (
      • Cheatham B.
      • Kahn C.R.
      ,
      • Fry M.J.
      ) and the other by the small GTP-binding protein p21ras (
      • Cheatham B.
      • Kahn C.R.
      ). The major route by which insulin stimulates PI 3-kinase activity is through tyrosine phosphorylation of IRS-1, as described above. Insulin stimulates Ras activity through a dual mechanism involving Shc, and to a lesser extent, IRS-1, which subsequently binds to the Grb2·SOS complex, activating p21ras(
      • Sasaoka T.
      • Draznin B.
      • Leitner J.W.
      • Langlois J.W.
      • Olefsky J.M.
      ). Stimulated Ras then activates a cascade of protein serine/threonine kinases which includes Raf, MEK, and the mitogen-activated protein (MAP) kinases, ERK1 and ERK2 (reviewed in Refs.
      • Cheatham B.
      • Kahn C.R.
      ,
      • Myers Jr., M.G.
      • White M.F.
      , and
      • Waters S.B.
      • Pessin J.E.
      ). Activation of both of these pathways appears to be necessary for the mitogenic effects of insulin (
      • Sasaoka T.
      • Draznin B.
      • Leitner J.W.
      • Langlois J.W.
      • Olefsky J.M.
      ,
      • Jhun B.H.
      • Meinkoth J.L.
      • Leitner J.W.
      • Draznin B.
      • Olefsky J.M.
      ,
      • Ullrich A.
      • Schlessinger J.
      ), whereas the metabolic effects of insulin are primarily activated by PI 3-kinase-dependent steps (
      • Kaburagi Y.
      • Satoh S.
      • Tamemoto H.
      • Yamamoto-Honda R.
      • Tobe K.
      • Veki K.
      • Yamauchi T.
      • Kono-Sugita E.
      • Sekihara H.
      • Aizawa S.
      • Cushman S.W.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Krook A.
      • Moller D.E.
      • Dib K.
      • O'Rahilly S.
      ). Thus, it has been shown that activated PI 3-kinase is both necessary and sufficient for insulin-stimulated GLUT4 translocation and glucose uptake (
      • Conricode K.M.
      ,
      • Margalet-Sanchez V.
      • Goldfine I.D.
      • Vlahos C.J.
      • Sung C.K.
      ,
      • Berger J.
      • Hayes N.
      • Szalkowksi D.M.
      • Zhang B.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ), and downstream targets of PI 3-kinase such as AKT and p70 S6 kinase may play important roles in the ultimate metabolic actions of this hormone (
      • Tanti J.-F.
      • Grillo S.
      • Gremeaux T.
      • Coffer P.J.
      • Van Obberghen E.
      • Marchand-Brustel Y.L.
      ,
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ).
      By no means are the PI 3-kinase and Ras/MAP kinase pathways completely separate. For example, Yamauchi et al. (
      • Yamauchi K.
      • Holt K.
      • Pessin J.E.
      ) have demonstrated that PI 3-kinase may lie upstream of Ras and Raf in mediating mitogenic effects of insulin. On the other hand, experiments using wortmannin to inhibit PI 3-kinase activity have shown that the effect of insulin on p21ras·GTP loading in CHO cells is independent of PI 3-kinase (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ). However, reciprocal relationships between PI 3-kinase and p21ras have also been demonstrated (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). Thus, one report has shown that activated Ras can stimulate PI 3-kinase activity, whereas other investigators have shown that the biologic effects of constitutively activated PI 3-kinase are Ras-dependent (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.W.
      ). Thus, although the exact relationship between PI 3-kinase and p21ras is unclear, abundant data exist to indicate that they are interconnected in some way (
      • Sjolander A.
      • Yamamoto K.
      • Huber B.E.
      • Lapetina E.G.
      ).
      In the current studies, we have extensively evaluated the role of PI 3-kinase activity in a diverse array of insulin actions. Our approach was based upon the notion that cellular overexpression of the N-SH2 domain of the p85 subunit would disrupt complex formation between endogenous PI 3-kinase and its SH2 domain binding targets, such as IRS-1. This, in turn, would prevent insulin activation of PI 3-kinase enzymatic activity, allowing us to elucidate the biologic actions of this enzyme in insulin target cells such as 3T3-L1 adipocytes. Since gene transfer into 3T3-L1 adipocytes can be problematic, we elected to use recombinant adenovirus to achieve these goals. We (
      • Sharma P.M.
      • Egawa K.
      • Gustafson T.A.
      • Martin J.L.
      • Olefsky J.M.
      ), and others (
      • Frevert E.U.
      • Khan B.B.
      ), have recently demonstrated that adenovirus mediated gene delivery allows high efficiency gene transduction and protein expression in terminally differentiated 3T3-L1 adipocytes. Thus, we prepared a recombinant adenovirus encoding the N-SH2 domain of the p85 α subunit of PI 3-kinase in order to overexpress this protein domain within 3T3-L1 adipocytes. Using this approach, we have investigated the acute effects of interrupting p85 association with insulin-stimulated target proteins on the ultimate biologic effects of insulin.

      DISCUSSION

      Insulin stimulation leads to extensive tyrosine phosphorylation of IRS-1. Phosphorylated IRS-1 binds to the SH2 domains of the p85 subunit of PI 3-kinase and activates its p110 subunit, and this is a major mechanism by which insulin stimulates this enzyme. Several lines of evidence suggest that PI 3-kinase is involved in mediating mitogenic effects of various growth factors (
      • Braselmann S.
      • Palmer T.M.
      • Cook S.J.
      ,
      • Myers Jr., M.G.
      • White M.F.
      ,
      • Kapeller R.
      • Cantley L.C.
      ) and is a necessary molecule for insulin stimulation of glucose transport (
      • Conricode K.M.
      ,
      • Margalet-Sanchez V.
      • Goldfine I.D.
      • Vlahos C.J.
      • Sung C.K.
      ,
      • Berger J.
      • Hayes N.
      • Szalkowksi D.M.
      • Zhang B.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ). Despite a great deal of information on both the structure and function of PI 3-kinase, the precise mechanism of its involvement in mediating insulin signaling is not fully understood.
      In the current study, we have extensively evaluated the role of PI 3-kinase activity in a diverse array of insulin actions. Our approach is based on the hypothesis that cellular overexpression of the N-SH2 domain of the p85 subunit would competitively inhibit complex formation between endogenous PI 3-kinase and its SH2 domain binding targets such as IRS-1, -2, -3, -4, the insulin receptor, and others. This in turn, will prevent insulin activation of PI 3-kinase enzymatic activity, allowing us to elucidate the biologic actions of this enzyme in insulin target cells such as 3T3-L1 adipocytes. Indeed our data show not only a marked inhibition of insulin-stimulated IRS-1-associated PI 3-kinase activity, but we find a complete blockade of insulin-stimulated anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. Predictably, and in agreement with previous findings, inhibition of PI 3-kinase activity resulted in abrogation of insulin's effects on glucose uptake and DNA synthesis (
      • Sasaoka T.
      • Draznin B.
      • Leitner J.W.
      • Langlois J.W.
      • Olefsky J.M.
      ,
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ,
      • Jhun B.H.
      • Meinkoth J.L.
      • Leitner J.W.
      • Draznin B.
      • Olefsky J.M.
      ,
      • Ullrich A.
      • Schlessinger J.
      ,
      • Kaburagi Y.
      • Satoh S.
      • Tamemoto H.
      • Yamamoto-Honda R.
      • Tobe K.
      • Veki K.
      • Yamauchi T.
      • Kono-Sugita E.
      • Sekihara H.
      • Aizawa S.
      • Cushman S.W.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Krook A.
      • Moller D.E.
      • Dib K.
      • O'Rahilly S.
      ). Other major findings of these experiments are that inhibition of PI 3-kinase activity resulted in inhibition of glycogen synthase activity and stimulation of MAPK activation by insulin. In contrast, overexpression of the p85N-SH2 protein led to a 2-fold increase in basal, insulin-independent GTP-bound levels of p21ras. However, insulin treatment did not further alter the levels of GTP-bound p21ras in these cells. Inhibition of the PI 3-kinase activity, also led to significant impairment of other downstream signaling effects of insulin such as Akt and p70 S6 kinase phosphorylation. These results lead to several predictions and conclusions.
      The activation of MAPK by insulin depends on activation of Ras (
      • Cheatham B.
      • Kahn C.R.
      ,
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ). Tyrosine-phosphorylated Shc (
      • Sasaoka T.
      • Draznin B.
      • Leitner J.W.
      • Langlois J.W.
      • Olefsky J.M.
      ), and to a lesser extent IRS-1 (
      • Cheatham B.
      • Kahn C.R.
      ,
      • Myers Jr., M.G.
      • White M.F.
      ,
      • Waters S.B.
      • Pessin J.E.
      ), binds to the adaptor protein, Grb-2, which is preassociated with the guanine nucleotide exchange factor, Sos, that promotes the formation of the active GTP-bound state of Ras. Formation of GTP-bound Ras leads to activation of the protein serine/threonine kinase cascade including Raf-1 kinase, MEK and MAPK (
      • Cheatham B.
      • Kahn C.R.
      ,
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ). Recent studies using specific inhibitors of PI 3-kinase or constitutively active and dominant negative mutants of the enzyme, have yielded confusing, and potentially conflicting results suggesting that PI 3-kinase can be either downstream or upstream of Ras (
      • Yamauchi K.
      • Holt K.
      • Pessin J.E.
      ,
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.W.
      ,
      • Sjolander A.
      • Yamamoto K.
      • Huber B.E.
      • Lapetina E.G.
      ). In the present study, using 3T3-L1 adipocytes, we have demonstrated that inhibition of PI 3-kinase decreased insulin-induced activation of MAPK, although the levels of GTP-bound Ras were increased ∼2-fold in the basal state. Thus, PI 3-kinase may be required for the activation of MAPK at a step independent of and downstream of Ras.
      The importance of PI 3-kinase for activation of ERKs 1 and 2 has been controversial. Expression of activated forms of p110α has been reported to stimulate the MAPK pathway in one case (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.W.
      ), but not in others (
      • Frevert E.U.
      • Khan B.B.
      ,
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M-A.
      • Williams L.W.
      ,
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evans G.
      ,
      • Lopez-Ilasaca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ). A recent study found that overexpression of the p110γ isoform resulted in activation of ERKs 1 and 2, whereas p110α was without effect (
      • Lopez-Ilasaca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ). Our findings indicating the involvement of PI 3-kinase in the activation of MAPK seem to be consistent with a recent report showing that treatment of 3T3-L1 adipocytes, either with wortmannin or LY294002, inhibited insulin-induced activation of Raf-1 and MAPKs with no effect on the formation of GTP-bound Ras (
      • Suga J.
      • Yoshimasa Y.
      • Yamada K.
      • Yamamoto Y.
      • Inoue G.
      • Okamoto M.
      • Hayashi T.
      • Shigemoto M.
      • Kosaki A.
      • Kuzuya H.
      • Nakao K.
      ). However, wortmannin did not affect epidermal growth factor-induced activation of Raf and MAPK in the same system, indicating that differential mechanisms to activate Raf-1 and MAPKs by insulin and epidermal growth factor exist in 3T3-L1 adipocytes. Very similar observations have been reported using rat adipocytes (
      • Liu H.
      • Lee J.
      • Kublaoui B.
      • Pilch P.F.
      ). However, the involvement of PI 3-kinase in MAPK activation appears to be cell type-specific, because wortmannin did not inhibit insulin-induced activation of MAPK in CHO cells in the above study (
      • Suga J.
      • Yoshimasa Y.
      • Yamada K.
      • Yamamoto Y.
      • Inoue G.
      • Okamoto M.
      • Hayashi T.
      • Shigemoto M.
      • Kosaki A.
      • Kuzuya H.
      • Nakao K.
      ).
      As mentioned earlier, the relationship between PI 3-kinase and Ras is a matter of debate. Some observations indicate that PI 3-kinase could be upstream, downstream, or independent of Ras; these observations are perhaps related to cell-type differences (reviewed in Ref.
      • Carpenter C.L.
      • Cantley L.C.
      ). Our data reinforce the idea that PI 3-kinase is upstream and can activate the Ras pathway. However, our unexpected findings were the influence of overexpression of the p85N-SH2 domain of PI 3-kinase to stimulate formation of p21ras·GTP in 3T3-L1 adipocytes. PI 3-kinase and Ras form a complex, suggesting an intimate relation between the function of these molecules (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ), and it can be argued that a loss of PI 3-kinase activity could disrupt basal Ras-p110 binding, which may be required for PI 3-kinase signaling.
      It is known that insulin stimulates p21ras·GTP loading by increasing the guanine nucleotide exchange activity of Sos (
      • Draznin B.
      • Chang L.
      • Leitner J.W.
      • Takata Y.
      • Olefsky J.M.
      ,
      • Medema R.H.
      • deVries-Smits A.M.M.
      • van der Zon G.C. M,.
      • Maassen J.A
      • Bos J.L.
      ). Return of p21ras into its inactive conformation is facilitated by GAP that hydrolyzes GTP to GDP. Therefore, overexpression of the p85N-SH2 domain of PI 3-kinase can stimulate formation of p21ras·GTP either by stimulating the guanine exchange activity of Sos or by diminishing the activity of GAP. Stimulation of Sos activity is unlikely because Langolis et al. (
      • Langolis J.
      • Leitner W.
      • Medth T.
      • Sasaoka T.
      • Olefsky J.M.
      • Draznin B.
      ) have shown that immunodepletion of PI 3-kinase from lysates of insulin-stimulated cells does not alter the activity level of the guanine nucleotide exchange factor Sos. Therefore, the most likely explanation for the increase in basal GTP-bound Ras in the p85N-SH2 expressing cells is that the p85N-SH2 depletes the pool of GAP in these cells. Indeed, in 3T3-L1 adipocytes, the p85 subunit of PI 3-kinase is already in excess compared with the p110 subunit, and it has been shown to interact with GAP (presumably via p62, a GAP-associated protein) (
      • DePaolo D.
      • Reusch J.E.-B.
      • Carel K.
      • Bhuripanyo P.
      • Leitner J.W.
      • Draznin B.
      ). Overexpression of the p85N-SH2 peptide could titrate out cellular GAP by direct association, such that GAP is not available to hydrolyze p21ras·GTP. The elevated p21ras·GTP levels do not appear to affect MAPK activity, since we find that the p85N-SH2 protein inhibits insulin-induced MAPK phosphorylation in 3T3-L1 adipocytes. Therefore, the inhibitory effect of PI 3-kinase on the MAPK cascade most likely occurs at a level independent of Ras. Therefore, in these cells, insulin is able to stimulate MAPK via a Ras-independent pathway. These results are in agreement with the observations of Carel et al. (
      • Carel K.
      • Kummer J.L.
      • Schubert C.
      • Leitner J.W.
      • Heidenreich K.A.
      • Draznin B.
      ), who also found that insulin activates MAPK by a Ras-independent pathway in 3T3-L1 adipocytes and by a Ras-dependent pathway in 3T3-L1 fibroblasts.
      In an earlier report, we utilized an adenovirus system to express the PTB domain of IRS-1 in 3T3-L1 adipocytes. This led to a marked decrease in IRS-1-associated PI 3-kinase activity, but no inhibition of insulin-stimulated glucose transport (
      • Sharma P.M.
      • Egawa K.
      • Gustafson T.A.
      • Martin J.L.
      • Olefsky J.M.
      ). The current study further extends our earlier proposed hypothesis that complete blockade of PI 3-kinase activity (i.e. IRS-1 and the non-IRS-1-associated components) would be necessary to inhibit insulin-induced glucose transport. Indeed, we find that this is the case from these results. As shown in Fig. 5 B, the dominant negative p85 completely blocks PI 3-kinase activity in the phosphotyrosine immunoprecipitates. The number of insulin receptor substrates is expanding rapidly, with the addition of IRS-2, -3, and -4 to the earlier insulin-substrates: IRS-1 and Shc. Among these, IRS-2 and IRS-3 (pp60) are tyrosine phosphorylated by insulin and bind to PI 3-kinase (
      • Lavan B.E.
      • Lane W.S.
      • Lienhard G.E.
      ,
      • Tobe K.
      • Tamemoto H.
      • Yamauchi T.
      • Aizawa S.
      • Yazaki Y.
      • Kadowaki T.
      ). It is possible that insulin stimulates glucose transport by at least two parallel pathways, which may be interacting or redundant. The current data support the notion that inhibition of glucose transport results from blockade of a yet to be identified, non-IRS-1-associated PI 3-kinase activity. Consistent with this, studies by Kaburagi et al. (
      • Kaburagi Y.
      • Satoh S.
      • Tamemoto H.
      • Yamamoto-Honda R.
      • Tobe K.
      • Veki K.
      • Yamauchi T.
      • Kono-Sugita E.
      • Sekihara H.
      • Aizawa S.
      • Cushman S.W.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ) on fat cells derived from IRS-1 knockout mice, and in which IRS-2 is negligible, show only a 50% reduction in maximal insulin-stimulated glucose transport, indicating the existence of an IRS-1/2-independent, but PI 3-kinase-dependent pathway for transport stimulation (
      • Kaburagi Y.
      • Satoh S.
      • Tamemoto H.
      • Yamamoto-Honda R.
      • Tobe K.
      • Veki K.
      • Yamauchi T.
      • Kono-Sugita E.
      • Sekihara H.
      • Aizawa S.
      • Cushman S.W.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ). In addition, studies by Krook et al. (
      • Krook A.
      • Moller D.E.
      • Dib K.
      • O'Rahilly S.
      ) and Isakoff et al. (
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ) are also consistent with the notion that IRS-1 phosphorylation, and its associated PI 3-kinase activation, are not sufficient to initiate metabolic signaling, suggesting that additional insulin-derived metabolic inputs must exist.
      We also examined additional targets of insulin action which are thought to be downstream of PI 3-kinase, to further explore the effects of PI 3-kinase inhibition. Akt, a serine/threonine kinase, is a downstream target of PI 3-kinase and is activated by a dual mechanism involving the binding of phosphatidylinositol 3,4-bisphosphate to its PH domain, as well as serine/threonine phosphorylation by one or more Akt kinases, which may themselves be stimulated by the lipid products of PI 3-kinase (
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.J.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ). Expression of a constitutively active Akt in rat adipocytes (
      • Tanti J.-F.
      • Gremeaux T.
      • Grillo S.
      • Calleja V.
      • Klippel A.
      • Williams L.T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ) and 3T3-L1 adipocytes (
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ) increases glucose uptake and GLUT4 translocation. Consistent with these findings, our data show that Akt activation is dependent on PI 3-kinase activity, since we find that pretreatment of 3T3-L1 adipocytes with wortmannin, a PI 3-kinase inhibitor, as well as overexpression of the dominant inhibitory p85N-SH2 protein, leads to an inhibition of insulin-induced serine/threonine kinase activity of Akt. In addition, insulin-induced glucose transport and glycogen synthase were inhibited when 3T3-L1 adipocytes were pretreated with wortmannin, or when the p85N-SH2 protein was overexpressed. Similarly, insulin-induced phosphorylation of another downstream target of PI 3-kinase, p70 S6 kinase, was inhibited by Rapamycin as well as by the dominant inhibitory p85N-SH2 protein. These results suggest a role for Akt as a PI 3-kinase-dependent upstream activator of glucose transport and/or a role for p70 S6 kinase as a PI 3-kinase-dependent upstream activator of glycogen synthase activity.
      Our data on expression of the membrane-targeted p110CAAX indicate that membrane localization of PI 3-kinase is critical for biologic functions, since this form of the p110 subunit leads to stimulation of 2-deoxyglucose transport similar to the insulin induced levels in 3T3-L1 adipocytes. Along similar lines, it has been shown in COS-7 cells, that targeting p110 to the membrane, either by N-terminal myristoylation or by C-terminal farnesylation, can stimulate p70 S6 kinase and Akt activity in the absence of insulin. Our results on co-expression of the p110CAAX with the N-SH2 domain strongly support the idea that the p85N-SH2 protein specifically blocks insulin mediated PI 3-kinase activity, by competitively interfering with the targeting of p85 to its natural protein partners. Thus, although the functional activity of downstream signaling molecules, such as p70 S6 kinase, Akt, and GLUT4, are functionally blocked by expression of the p85N-SH2, the fact that the biologic activity can be rescued by co-expression of the N-SH2 domain with p110CAAX demonstrates that the signaling pathways downstream of p85 targeting are intact and that the dominant/negative effects of the p85N-SH2 domain do not reflect nonspecific actions at other cellular loci.
      Our studies also show that overexpression of the p85N-SH2 domain markedly inhibits insulin-stimulated glycogen synthase activity. This leads to the conclusion that PI 3-kinase stimulation is necessary for insulin activation of glycogen synthase. The molecular mechanisms by which glycogen synthase is regulated by insulin remains one of the crucial issues in insulin action. Insulin stimulates glycogen synthesis via a coordinated response involving activation of protein phosphatase 1, by phosphorylation of its G subunit and by a phosphorylation-induced inactivation of glycogen synthase kinase 3 (GSK3) (
      • Parker P.J.
      • Caudwell F.B.
      • Cohen P.
      ,
      • Shulman R.G.
      • Bloch G.
      • Rothman D.L.
      ). It has been suggested that pp1 might be downstream of the MAPK pathway, but a number of reports have indicated that this is not the case (
      • Lawrence J.C.
      • Roach P.J.
      ). In addition, our own results show that the MEK inhibitor (PD098059) did not lead to a decrease in insulin stimulation of glycogen synthesis. Thus, it is unlikely that any effect of the p85N-SH2 protein on the MAPK pathway is mediating the inhibitory effect on glycogen synthesis. It has been reported that GSK3 is a downstream target of Akt, which, in turn, is dependent on PI 3-kinase activity (
      • Cross D.A.E.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.
      ). Thus, one potential interpretation of our results is that inhibition of PI 3-kinase, through the dominant/negative p85N-SH2 domain, prevents insulin-stimulated GSK3 phosphorylation, and that this leads to inhibition of glycogen synthesis. However, it has recently been shown that GSK3 expression is either very low, or not expressed, in 3T3-L1 adipocytes (
      • Benjamin W.B.
      • Pentyala S.N.
      • Woodgett J.R.
      • Hod Y.
      • Marshak D.
      ,
      • Cross D.A.E.
      • Watt P.W.
      • Shaw M.
      • van der Kaay J.
      • Downes C.P.
      • Holder J.C.
      • Cohen P.
      ,
      • Moule S.K.
      • Welsh G.I.
      • Edgell N.J.
      • Foulstone E.J.
      • Proud C.G.
      • Denton R.M.
      ), making this possibility less likely. Thus, although the exact mechanism is unknown, our results clearly show that insulin-stimulated glycogen synthesis is dependent on proper targeting of PI 3-kinase, and this is consistent with other reports showing that insulin-stimulated glycogen synthesis can be inhibited by wortmannin or LY294002 (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Shepherd P.R.
      • Nave B.T.
      • Siddle K.
      ), both of which are relatively specific inhibitors of PI 3-kinase activity.
      In conclusion, we have shown that overexpression of the p85N-SH2 domain in 3T3-L1 adipocytes inhibits a variety of insulin's ultimate biologic effects, and that this domain will also block insulin-stimulated DNA synthesis in fibroblasts. These results are consistent with our earlier findings, which suggested that a complete blockade of PI 3-kinase targeting to all of its cellular substrates (IRS-1 as well as non-IRS-1 targets) would be necessary for inhibition of AKT activation and glucose transport stimulation. We also find that in 3T3-L1 adipocytes, PI 3-kinase can regulate p21ras·GTP levels and that the dominant/negative effects of the p85 NSH2 domain on the MAP kinase pathway are exerted distal to the stimulatory effect of p21ras·GTP on the MAPK pathway.

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