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Phosphatidylinositol-3 Kinase Is Necessary for 12-O-Tetradecanoylphorbol-13-acetate-induced Cell Transformation and Activated Protein 1 Activation*

Open AccessPublished:February 14, 1997DOI:https://doi.org/10.1074/jbc.272.7.4187
      Phorbol esters, which activate isoforms of protein kinase C, are general activators of the transcription factor activated protein 1 (AP-1). The pathway involved in this signal transduction is not very clear. Currently, little is known about whether phosphatidylinositol-3 (PI-3) kinase plays any role in phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced signal transduction. We demonstrate here that TPA not only has markedly synergistic effects on insulin-induced PI-3 kinase activity, but it also can induce PI-3 kinase activity and the PI-3 phosphates by itself. We also found that insulin, a PI-3 kinase activator, enhanced TPA-induced AP-1 trans-activation and transformation in JB6 promotion-sensitive cells. Furthermore, wortmannin and LY294002, two PI-3 kinase inhibitors, markedly decreased AP-1 activity induced by insulin, TPA, or TPA and insulin and inhibited JB6 promotion-sensitive cell transformation induced by TPA or TPA and insulin. Most importantly, constitutive overexpression of the dominant negative PI-3 kinase P85 mutants completely blocked insulin- or TPA-induced AP-1 trans-activation and TPA-induced cell transformation. All evidence from present studies suggests that PI-3 kinase acts as a mediator in TPA-induced AP-1 activation and transformation in JB6 cells.

      INTRODUCTION

      Phosphatidylinositol-3
      The abbreviations used are: PI
      phosphatidylinositol
      AP-1
      activated protein 1
      FBS
      fetal bovine serum
      IGF-IR
      insulin-like growth factor I receptor
      IR
      insulin receptor
      MAP
      mitogen-activated protein
      MEM
      minimal essential medium
      P+
      promotion-sensitive
      PI-3,4-P2
      phosphatidylinositol-3,4-bisphosphate
      PKC
      protein kinase C
      TPA
      12-O-tetradecanoylphorbol-13-acetate
      BME
      basal medium Eagle.
      (PI-3) kinase plays a central role in a broad range of biological effects (
      • Cantley L.C.
      • Auger K.R.
      • Carpetner C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ). This enzyme is a dimer composed of a catalytic subunit (P110) and a regulatory subunit (P85) (
      • Carpenter C.L.
      • Duckworth B.C.
      • Auger K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ). The P85 regulatory subunit has no discernible catalytic activity but possesses two Src homology 2 domains and an Src homology 3 domain (
      • Kapeller R.
      • Cantley L.C.
      ). A region between the two Src homology 2 domains of P85 binds the NH2 terminus of P110, mediating the constitutive association of the two subunits (
      • Kapeller R.
      • Cantley L.C.
      ). Binding of P85 to P110 partially activates P110 (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ,
      • Hu P.
      • Schlessinger J.
      ). PI-3 kinase phosphorylates the lipid PI on the 3 position of the D-myoinositol ring, yielding PI-3-phosphate (
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ). Because the enzyme can also use phosphorylated forms of phosphatidylinositol (PI-4-phosphate and PI-4,5-bisphosphate) as substrates, activation of the PI-3 kinase also leads to the generation of PI-3,4-P2 and PI-3,4,5-triphosphate (
      • Carpenter C.L.
      • Duckworth B.C.
      • Auger K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ,
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ,
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ). Previous studies suggested that these PI-3 kinase products are potential second messengers (
      • Cantley L.C.
      • Auger K.R.
      • Carpetner C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Downes C.P.
      • Carter A.N.
      ,
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      ).
      12-O-Tetradecanoylphorbol-13-acetate (TPA) is not only a potent tumor promoter in mouse skin (
      • Berenblum I.
      • Becker F.F.
      Cancer, A Comprehensive Treatise.
      ,
      • Slaga T.J.
      • Fischer S.M.
      • Weeks C.E.
      • Klein-Szanto A.J.P.
      • Reiners J.
      ), but it also induces a wide range of other biological effects in cultured cells (
      • Diamong L.
      • O'Brien T.
      • Braid W.M.
      ). Protein kinase C (PKC) is well known as a TPA receptor and a phospholipid-dependent kinase involved in basic cellular functions, including regulation of cell growth, differentiation, and gene expression (
      • Blumberg P.M.
      ,
      • Arnold R.S.
      • Newton A.C.
      ). PKC isozymes include more than 11 different enzymes (α, βI, βII, γ, δ, ∊, ζ, η, θ, Λ, and μ). Previous studies indicated that the TPA-induced activation of AP-1, NFκB, and other transcription factors in the nucleus is mediated by the Ras-Raf-1/MAP kinase cascade (
      • Arnold R.S.
      • Newton A.C.
      ,
      • Schaap D.
      • van der Wal J.
      • Howe L.R.
      • Marshall C.J.
      • van Blitterswijk W.J.
      ,
      • Troppmair J.
      • Bruder J.T.
      • Munoz H.
      • Lloyd P.A.
      • Kyriakis J.
      • Banerjee P.
      • Avruch J.
      • Rapp U.R.
      ). The role of PI-3 kinase in the TPA-induced signal transduction pathway, however, is not clear, even though some reports indicated that PI-3 kinase and p21ras modulate each other's signals (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). For example, overexpression of activated p21ras in PC12 cells increases PI-3 kinase activity and stimulates the accumulation of 3′-phosphorylated inositol lipids in the cells (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). GTP-bound p21ras also directly binds and activates PI-3 kinase in vitro (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). Expression of activated PI-3 kinase in NIH 3T3 cells apparently potentiates p21ras-dependent signaling events (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ). Furthermore, PI-3 kinase products may play an important role in extensive cross-talk among multiple signaling pathways and regulation of cell function (
      • Cantley L.C.
      • Auger K.R.
      • Carpetner C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Downes C.P.
      • Carter A.N.
      ,
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      ). Since both phorbol ester- and insulin-induced activation of the Ras-Raf/MAP kinase pathway and a combination of insulin and phorbol ester resulted in a synergistic activation of this pathway (
      • Knoepp S.M.
      • Wisehart-Johnson A.E.
      • Buse M.G.
      • Bradshaw C.D.
      • Ella K.M.
      • Meier K.E.
      ), we inquired whether TPA can activate PI-3 kinase and whether activation of PI-3 kinase is required for TPA-induced signal transduction and cell transformation. In the present study we used several approaches, which included a PI-3 kinase activator, two pharmacological PI-3 kinase inhibitors, and a dominant negative PI-3 kinase inhibitor, to study the role of PI-3 kinase in TPA-induced AP-1 activation and cell transformation in the well characterized mouse epidermal JB6 P+ (tumor promotion-sensitive) cells.

      DISCUSSION

      Our present studies demonstrate that PI-3 kinase is a crucial mediator of TPA-induced cell transformation and AP-1 trans-activation in JB6 cells. TPA alone could induce PI-3 kinase activity and increase the level of PI-3,4-P2 in JB6 cells. More interestingly, TPA and insulin synergistically induced PI-3 kinase activity. Insulin, a strong PI-3 kinase activator, enhanced TPA-induced AP-1 activation and cell transformation. Furthermore, wortmannin and LY294002, two different kinds of PI-3 kinase inhibitors, which inhibit PI-3 kinase by interfering with P110 and P85, respectively, inhibit TPA-induced AP-1 activation as well as cell transformation. More convincingly, TPA- and TPA- and insulin-induced AP-1 activation and TPA-induced cell transformation could be blocked completely by overexpression of the dominant negative PI-3 kinase P85 mutants in all time courses and dose responses studied. In contrast, we found previously that wortmannin or LY294002, as well as overexpression of δP85, had no significant inhibitory effect on JB6 cell proliferation and UV-induced AP-1 activity (
      • Huang C.
      • Ma W.
      • Dong Z.
      ). These observations suggest that the inhibition of AP-1 activation and cell transformation occurred through blocking PI-3 kinase activity but not because of any cytotoxic effect.
      Activation of PKC requires both association with the membrane and a number of activators and cofactors, the requirements for which differ for each isozyme (
      • Nishizuka Y.
      ). Thus, PKCs are grouped into three major classes: conventional PKC isoforms, such as α, βI, βII, and γ; novel PKCs, including δ, ∊, η, and θ; and atypical PKCs, represented by the ζ and Λ isozymes of PKC (
      • Nishizuka Y.
      ,
      • Hug H.
      • Sarre T.F.
      ). Activation of atypical PKCs could be carried out by either the PI-3 kinase pathway or the ceramide pathway (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Divecha N.
      • Irvine R.F.
      ). Conventional PKCs are activated by diacylglycerol in a Ca+-dependent manner. In contrast, activation of novel PKCs is Ca+-independent (
      • Nishizuka Y.
      ,
      • Hug H.
      • Sarre T.F.
      ). In addition to the natural activator, conventional PKCs and novel PKCs are also activated with high specificity by TPA (
      • Blumberg P.M.
      ). For this reason, TPA is often used in the study of the mechanisms of conventional PKC and novel PKC activation and their function. Most of the previous studies have addressed the regulation of phosphorylation of the insulin receptor by PKC in PKC isozyme-transfected cells (
      • Chin J.E.
      • Dickens M.
      • Tavare J.M.
      • Roth R.A.
      ,
      • Danielsen A.G.
      • Liu F.
      • Hosomi Y.
      • Shii K.
      • Roth R.A.
      ). Overexpression of PKC isozymes α, βI, γ, and ∊ did not affect expression of the insulin receptor or insulin-stimulated tyrosine phosphorylation of the receptor. However, in response to phorbol esters, cells expressing PKC α, γ, and βI but not ∊ exhibited 3-4-fold higher levels of insulin receptor (IR) phosphorylation. This TPA-stimulated IR phosphorylation inhibits the activation of the insulin receptor kinase and the insulin-induced PI-3 kinase activity as well as the tyrosine phosphorylation of Shc (
      • Chin J.E.
      • Dickens M.
      • Tavare J.M.
      • Roth R.A.
      ,
      • Danielsen A.G.
      • Liu F.
      • Hosomi Y.
      • Shii K.
      • Roth R.A.
      ), but this inhibition is not observed in the cells containing only the endogenous levels of PKC (
      • Danielsen A.G.
      • Liu F.
      • Hosomi Y.
      • Shii K.
      • Roth R.A.
      ). In the present study, we demonstrated that TPA induces a low level of PI-3 kinase activity and has significant synergistic effects with insulin on activation of PI-3 kinase in mouse epidermal JB6 cells. The reason for the difference among data from different cells may be due to different levels of endogenous PKC in the various cell types studied as well as differences in ratios of various PKC isozymes present in different cells. The ratio of PKC:IR in different cells may be another reason for these differences.
      Several studies suggested that the PI-3 kinase and its products PI-3,4-P2 and PI-3,4,5-triphosphate are important regulators of cell proliferation and c-fos gene expression (
      • Cantley L.C.
      • Auger K.R.
      • Carpetner C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ). The introduction of the NH2-terminal Src homology 2 domain of the P85 subunit of PI-3 kinase into cells abrogates the insulin- or IGF-1-stimulated DNA synthesis and prevents insulin stimulation of c-Fos protein expression (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ). The microinjection of a dominant-negative p21ras mutant or anti-Ras antibody inhibited insulin-induced DNA synthesis (
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ). A constitutively activated mutant P110 induced transcription from the Fos promoter; coexpression of dominant negative Ras blocked this response (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ). Other studies have shown that PI-3,4-P2 and PI-3,4,5-triphosphate are elevated in cells transformed by v-abl, v-src, and polyoma middle T, and decreased levels of these lipids correlate with impaired cell transformation by mutated forms of these oncogenes (
      • Cantley L.C.
      • Auger K.R.
      • Carpetner C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Fukui Y.
      • Saltiel A.R.
      • Hanafusa H.
      ,
      • Varticovski L.
      • Daley G.Q.
      • Jackson P.
      • Baltimore D.
      • Cantley L.C.
      ). It has been reported that the presence of insulin-like growth factor I receptor (IGF-IR) is an obligatory requirement for the establishment and maintenance of the tumor phenotype (
      • Baserga R.
      ,
      • Coppola D.
      • Ferberg A.
      • Miura M.
      • Sell C.
      • D'Ambrosio C.
      • Rubin R.
      • Baserga R.
      ,
      • Miura M.
      • Surmacz E.
      • Burgaud J.L.
      • Baserga R.
      ,
      • Surmacz E.
      • Sell C.
      • Swartek J.
      • Kato H.
      • Roberts C.T.
      • Leroith D.
      • Baserga R.
      ). Cells derived from mouse embryos with a targeted disruption of the IGF-IR gene (R cells) cannot be transformed by SV40 T antigen or by an activated and overexpressed Ha-ras, even by a combination of both, all of which transform very efficiently the corresponding wild type cells or other 3T3-like cells (
      • Sell C.
      • Rubini M.
      • Rubin R.
      • Liu J.-P.
      • Etstratiadis A.
      • Baserga R.
      ). If a plasmid expressing a wild type human IGF-I receptor cDNA is stably transfected into R cells, the cells can be transformed by SV40 T antigen. This indicates that the defect in transformability is specifically due to the lack of IGF-IR (
      • Surmacz E.
      • Sell C.
      • Swartek J.
      • Kato H.
      • Roberts C.T.
      • Leroith D.
      • Baserga R.
      ). Substantial evidence has been reported that PI-3 kinase is a critical component of signaling pathways used by the cell surface receptors for a variety of mammalian growth factors or other stimulators (
      • Cantley L.C.
      • Auger K.R.
      • Carpetner C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ,
      • Downes C.P.
      • Carter A.N.
      ,
      • Hawkins P.T.
      • Jackson T.R.
      • Stephens L.R.
      ), especially IR and IGF-IR. Recently, it was reported that insulin could activate the Ras-Raf/MAP kinase pathway by interacting and activating its receptors (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ). Dhand et al. (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ) suggested that activation of this Ras/MAP kinase pathway is critical for the effect of insulin on mitogenesis and c-fos expression. Others found that neither insulin nor phorbol ester regulation of phosphoenolpyruvate carboxykinase gene expression requires activation of the Ras/MAP kinase pathway, but PI-3 kinase is required in this event (
      • Gabbay R.A.
      • Sutherland C.
      • Gnudi L.
      • Kahn B.B.
      • O'Brien R.M.
      • Granner D.K.
      • Flier J.S.
      ). In contrast, Sakaue et al. (
      • Sakaue H.
      • Hara K.
      • Noguchi T.
      • Matozaki T.
      • Kotani K.
      • Ogawa W.
      • Yonezawa K.
      • Waterfield M.D.
      • Kasuga M.
      ) demonstrated that neither the Ras/MAP kinase cascade nor PI-3 kinase may be required for insulin-stimulated glycogen synthase activation in Chinese hamster ovary cell lines. Evidence from different groups using different models has confirmed the crucial importance of AP-1 activity in transformation and carcinogenesis (
      • Dong Z.G.
      • Birrer M.J.
      • Watts R.G.
      • Matrisian L.M.
      • Colburn N.H.
      ,
      • Huang C.S.
      • Ma W.Y.
      • Dong Z.G.
      ,
      • Alani R.
      • Brown P.
      • Binetruy B.
      • Dosaker H.
      • Rosenberg R.K.
      • Angel P.
      • Karin M.
      • Birrer M.J.
      ,
      • Dong Z.G.
      • Lavrovsky V.
      • Colburn N.H.
      ). Our previous results have shown that AP-1 activation is required for tumor promotion in the JB6 cell model (
      • Dong Z.G.
      • Birrer M.J.
      • Watts R.G.
      • Matrisian L.M.
      • Colburn N.H.
      ,
      • Huang C.S.
      • Ma W.Y.
      • Dong Z.G.
      ,
      • Domann F.E.
      • Levy J.P.
      • Birrer M.J.
      • Bowden G.T.
      ,
      • Dong Z.G.
      • Lavrovsky V.
      • Colburn N.H.
      ). High basal levels of AP-1 activity appear to be important for the maintenance of tumor phenotypes in the transformed cell line RT101 (
      • Domann F.E.
      • Levy J.P.
      • Birrer M.J.
      • Bowden G.T.
      ,
      • Dong Z.G.
      • Lavrovsky V.
      • Colburn N.H.
      ). Since our primary results (Fig. 1) showed that TPA can induce PI-3 kinase activity and increase the level of PI-3,4-P2 (Fig. 2), as well as have a markedly synergistic effect with insulin on PI-3 kinase activation, a critical question is whether PI-3 kinase plays any role in TPA-induced AP-1 activation and cell transformation. To test this hypothesis, we used several approaches. First, we treated cells with insulin, a very effective PI-3 kinase activator. The results showed that activation of PI-3 kinase by insulin resulted in a marked increase in TPA-induced AP-1 activity. Furthermore, we used two kinds of pharmacological PI-3 kinase inhibitors to block PI-3 kinase. The first inhibitor used in our study was a fungal metabolite, wortmannin, which covalently binds to the catalytic subunit P110 of PI-3 kinase and irreversibly inhibits the enzymatic activity at nanomolar concentrations (
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ). The second PI-3 kinase inhibitor used in this study was LY294002. Unlike wortmannin, LY294002 reversibly inhibits PI-3 kinase by competing with ATP for its substrate binding site (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ). These two inhibitors markedly inhibited AP-1 activation and cell transformation induced by TPA or TPA and insulin in a dose-dependent manner. Finally, we used a dominant negative mutant of PI-3 kinase, δP85, in our study. This dominant negative mutant has been shown to specifically block PI-3 kinase activity and its mediated cell function in intact cells (
      • Hare 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.
      ,
      • Sakaue H.
      • Hara K.
      • Noguchi T.
      • Matozaki T.
      • Kotani K.
      • Ogawa W.
      • Yonezawa K.
      • Waterfield M.D.
      • Kasuga M.
      ). The stable introduction of a dominant negative mutant of the PI-3 kinase P85 subunit (δP85) into JB6 cells was shown to block PI-3 kinase activity by insulin, TPA, or TPA and insulin and also to completely block TPA- or TPA- and insulin-induced AP-1 activity and cell transformation. All results from our experiments indicate that PI-3 kinase is necessary for TPA-stimulated AP-1 activation and cell transformation in JB6 cells. Recent reports suggest that PI-3 kinase and p21ras modulate each other's signals (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Hare 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.
      ). In JB6 cells, TPA alone induced a low level of PI-3 kinase activity. This TPA-induced low level of PI-3 kinase activity appears to be required for AP-1 activation and cell transformation. There are several models that may explain these data. One interpretation is that the cross-talk between PI-3 kinase and p21ras is important for the TPA-induced Ras-Raf/MAP kinase cascade leading to the AP-1 activation and cell transformation. Another interpretation is that there are some growth factors (such as IGF-I) that exist in the serum used in cell transformation and have a synergistic effect with TPA on induction of PI-3 kinase activity. The last possibility is supported by data from Fig. 5, in that the enhancement of TPA-induced AP-1 activity by insulin was only observed at a low concentration of serum.
      The signaling pathways induced by insulin have been the subject of intense research (
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ,
      • Knoepp S.M.
      • Wisehart-Johnson A.E.
      • Buse M.G.
      • Bradshaw C.D.
      • Ella K.M.
      • Meier K.E.
      ,
      • Hare 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.
      ,
      • Gabbay R.A.
      • Sutherland C.
      • Gnudi L.
      • Kahn B.B.
      • O'Brien R.M.
      • Granner D.K.
      • Flier J.S.
      ). Insulin is able to bind to both IR and IGF-IR, but affinities of these two receptors for insulin are different. The affinity of IR for insulin is at least 100 times higher than that of IGF-IR (
      • Schaffer L.
      ). Insulin binding to these two receptors results in receptor-mediated tyrosine phosphorylation of IRS-1 and Shc. These molecules then function as high affinity binding sites for the P85 subunit of PI-3 kinase, and this interaction subsequently results in the activation of PI-3 kinase. In the present studies, we used 2.5 μg/ml insulin as the optimal concentration of insulin for AP-1 activation and costimulation of cell transformation. In this concentration, insulin may bind to both IR or IGF-IR. To whatever receptor insulin binds, after activation of these receptors and subsequent activation of IRS-1, PI-3 kinase is the downstream target of IRS-1. Therefore, the data from our study, in which insulin enhanced the TPA-induced AP-1 activation and cell transformation, still support the concept that PI-3 kinase is required in TPA-induced AP-1 activation and cell transformation in JB6 cells.
      In conclusion, we have used several approaches to study the role of the PI-3 kinase in TPA-induced AP-1 activation and cell transformation in JB6 cells. TPA could induce PI-3 kinase, and this induction effect was synergistically enhanced by insulin. The two pharmacological inhibitors (wortmannin and LY294002) or the biological inhibitor (δP85, a dominant negative mutant of the PI-3 kinase P85 subunit of PI-3 kinase) markedly blocked TPA-induced AP-1 activation and cell transformation. Specific blockage of the events required for cell transformation with few side effects on normal growth might be a promising target for cancer prevention and treatment. In fact, inhibiting induced PI-3 kinase and AP-1 activity by a pharmacological inhibitor (wortmannin) or a dominant negative mutant of PI-3 kinase (δP85) does not seem to have inhibitory effects on cell growth in JB6 cells. Further investigation of this topic may elucidate the precise mechanisms underlying the role of PI-3 kinase in phorbol ester-induced signal transduction and may thus provide a novel target for the prevention of carcinogenesis.

      Acknowledgments

      We thank Dr. Masato Kasuga for the generous gift of the bovine PI-3 kinase P85 subunit mutant plasmid δP85, Dr. Vincent Duronio for supplying the protocol for HPLC analysis, and Jeanne Ruble for secretarial assistance.

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