SRF-dependent gene expression is required for PI3-kinase-regulated cell proliferation
2000; Springer Nature; Volume: 19; Issue: 18 Linguagem: Inglês
10.1093/emboj/19.18.4955
ISSN1460-2075
AutoresSteven Poser, Soren Impey, Kien Trinh, Zhengui Xia, Daniel R. Storm,
Tópico(s)Fungal Plant Pathogen Control
ResumoArticle15 September 2000free access SRF-dependent gene expression is required for PI3-kinase-regulated cell proliferation Steve Poser Steve Poser Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA, 98195 USA S.Poser and S.Impey contributed equally to this work Search for more papers by this author Soren Impey Soren Impey Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA, 98195 USA S.Poser and S.Impey contributed equally to this work Search for more papers by this author Kien Trinh Kien Trinh Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA, 98195 USA Search for more papers by this author Zhengui Xia Zhengui Xia Department of Environmental Health and Toxicology, School of Environmental Health, University of Washington, Seattle, WA, 98195 USA Search for more papers by this author Daniel R. Storm Corresponding Author Daniel R. Storm Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA, 98195 USA Search for more papers by this author Steve Poser Steve Poser Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA, 98195 USA S.Poser and S.Impey contributed equally to this work Search for more papers by this author Soren Impey Soren Impey Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA, 98195 USA S.Poser and S.Impey contributed equally to this work Search for more papers by this author Kien Trinh Kien Trinh Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA, 98195 USA Search for more papers by this author Zhengui Xia Zhengui Xia Department of Environmental Health and Toxicology, School of Environmental Health, University of Washington, Seattle, WA, 98195 USA Search for more papers by this author Daniel R. Storm Corresponding Author Daniel R. Storm Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA, 98195 USA Search for more papers by this author Author Information Steve Poser1,3, Soren Impey1,3, Kien Trinh1, Zhengui Xia2 and Daniel R. Storm 1 1Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA, 98195 USA 2Department of Environmental Health and Toxicology, School of Environmental Health, University of Washington, Seattle, WA, 98195 USA 3S.Poser and S.Impey contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4955-4966https://doi.org/10.1093/emboj/19.18.4955 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Recent evidence indicates that phosphatidylinositol 3-kinase (PI3K) is a central regulator of mitosis, apoptosis and oncogenesis. Nevertheless, the mechanisms by which PI3K regulates proliferation are not well characterized. Mitogens stimulate entry into the cell cycle by inducing the expression of immediate early genes (IEGs) that in turn trigger the expression of G1 cyclins. Here we describe a novel PI3K- regulated transcriptional cascade that is critical for mitogen regulation of the IEG, c-fos. We show that PI3K activates gene expression by transactivating SRF-dependent transcription independently of the previously described Rho and ETS TCF pathways. PI3K-stimulated cell cycle progression requires transactivation of SRF and expression of dominant- negative PI3K blocks mitogen-stimulated cell cycle progression. Furthermore, dominant-interfering SRF mutants attenuate mitogen-stimulated cell cycle progression, but are without effect on MEK-stimulated cell cycle entry. Moreover, expression of constitutively active SRF is sufficient for cell cycle entry. Thus, we delineate a novel SRF-dependent mitogenic cascade that is critical for PI3K- and growth factor-mediated cell cycle progression. Introduction Mitogens and growth factors activate phosphatidylinositol 3-kinase (PI3K) and promote the phosphorylation of phosphoinositides at the D3 position. These phospholipids play a critical role in the regulation of mitogenesis, apoptosis and cytoskeletal rearrangement (for a review see Rameh and Cantley, 1999). Type 1 PI3K is comprised of regulatory (p85) and catalytic (p110) subunits and was identified by virtue of its role in Src-mediated transformation (Sugimoto et al., 1984). Expression of constitutively active PI3K can trigger DNA synthesis (Frevert and Kahn, 1997; Klippel et al., 1998) and PI3K activity is required for mitosis in some cells (Jhun et al., 1994; Roche et al., 1994). Mice deficient for p110α catalytic subunits (Bi et al., 1999) have a proliferative defect that results in embryonic lethality. The identification of oncogenic forms of p110 and p85 PI3K further illustrates the critical role of PI3K in proliferation (Chang et al., 1997; Jimenez et al., 1998). The identification of the tumor suppressor, PTEN, as a D3 phosphoinositide phosphatase underscores the critical role of PI3K in cell growth and oncogenesis (Myers et al., 1998). A significant fraction of human cancers contain deletions or mutations of PTEN (for review see Cantley and Neel, 1999). However, the mechanisms by which PTEN regulates cell cycle progression are not clear and the targets of PI3K signaling during mitosis remain obscure. The ability of mitogens to trigger cell cycle progression depends on the rapid transcription of immediate early genes (IEGs). In particular, c-fos and related transcription factors play a critical role in mitogenesis (Riabowol et al., 1988; Kovary and Bravo, 1991) by inducing the expression of genes necessary for the activation of G1 cyclins (Brown et al., 1998). For example, cyclin D1 mRNA is increased by overexpression of c-fos (Miao and Curran, 1994) and mitosis and mitogen-stimulated cyclin D1 transcription are impaired in cells deficient for c-fos and FosB (Brown et al., 1998). Interestingly, mitogen-stimulated c-fos transcription requires PI3K activity (Yamauchi et al., 1993; Jhun et al., 1994) and activated PI3K stimulates c-fos transcription (Wang et al., 1998). The cis-acting serum response element (SRE) is crucial for mitogen-induced c-fos transcription and is important for the mitogen-stimulated transcription of many IEGs (Treisman, 1995). The MADS box-containing protein, serum response factor (SRF), binds to the SRE as a dimer and is necessary for the activation of SRE-mediated gene expression by mitogens (Norman et al., 1988). ETS family transcription factors (Elk1, Sap1a) bind a site adjacent to the SRE in the c-fos promoter and in some cells are required for growth factor-stimulated transcription (reviewed in Treisman, 1994). These ETS ternary complex factors (TCFs) are phosphorylated and transactivated by mitogen-activated protein kinases (MAPKs) (Treisman, 1995). Nevertheless, in many systems mitogens stimulate SRF-dependent transcription via an ETS-independent transcriptional pathway that targets the SRF DNA binding domain (Johansen and Prywes, 1994; Hill and Treisman, 1995). For example, in NIH 3T3 cells a Rho-dependent signaling pathway (Hill et al., 1995), a JNK-dependent histone acetylation cascade (Alberts et al., 1998) and the LIMK-1-dependent regulation of actin dynamics (Sotiropoulos et al., 1999) are required for ETS-independent transcription. The role of PI3K signaling in cell cycle progression and the ability of activated PI3K to promote entry into S phase led us to examine whether PI3K signaling regulates mitogen-stimulated c-fos expression. Although PI3K may induce c-fos transcription indirectly via crosstalk with Ras and Rho signaling cascades (Yamauchi et al., 1993; Urich et al., 1997; Wang et al., 1998), other studies suggest a novel mechanism (Jhun et al., 1994; Bruning et al., 1997; Montmayeur et al., 1997). Here we demonstrate that SRF mediates PI3K-regulated c-fos transcription and that activation of SRF-dependent transcription by PI3K occurs via a novel ETS TCF and Rho-independent pathway. We also show that PI3K-dependent cell cycle progression depends on activation of SRF-dependent gene expression. Results Growth factor-stimulated c-fos transcription requires PI3K signaling Because PI3K signaling regulates cell growth, we examined whether PI3K is required for growth factor-stimulated c-fos transcription. Treatment with nerve growth factor (NGF) increased c-fos transcription ∼6-fold in PC12 cells transfected with a c-fos promoter-regulated reporter construct (c-fos–LacZ) (Figure 1A). Treatment of cells with the PI3K inhibitor, wortmannin, or coexpression of a dominant-negative PI3K regulatory subunit (DN p85) significantly reduced NGF-mediated c-fos transcription. Mutations in the SRE (mSRE) that prevent SRF binding markedly attenuated NGF-stimulated c-fos transcription (Figure 1A). NGF-induced transcription of the endogenous c-fos gene also depended on PI3K signaling (Figure 1B). Figure 1.PI3K signaling is required for growth factor-stimulated c-fos transcription. (A) NGF stimulation of c-fos transcription required PI3K signaling through the SRE. PC12 cells were transfected with wild-type c-fos promoter–LacZ reporter construct (c-fos–LacZ) or a mutant SRE c-fos reporter construct (mSRE c-fos–LacZ). PI3K activity was blocked by cotransfection with a 3-fold excess (125 ng/well) of the dominant-negative regulatory subunit of PI3K (DN p85) DNA or pretreatment with 100 nM wortmannin (Wort). Cells were treated with 100 ng/ml NGF and assayed for LacZ activity. (B) Inhibition of PI3K attenuated NGF-induced c-fos transcription. PC12 cells were pretreated with 10 nM Wortmannin (Wort) or vehicle for 1 h and stimulated with 100 ng/ml NGF for 45 min. RT–PCR for c-fos was performed on total cellular RNA. β-actin transcription was used to normalize the level of c-fos transcription. (C) The PI3K signaling pathway was required for PDGF-mediated stimulation of c-fos expression. PC12 cells were transfected with the wild-type or mSRE c-fos LacZ reporter plasmids and either wild-type PDGFβ receptor (PDGFR wt), or mutants deficient in coupling to PI3K activation (Y741F/Y750F) or Shc/Ras GAP activation (Y771F) (375 ng/well). Cells were treated with 30 ng/ml PDGF and assayed for LacZ activity. The data are means ± standard deviation (SD) of triplicate assays normalized to luciferase activity driven from an SV40-luciferase plasmid. Download figure Download PowerPoint PDGFβ receptors (PDGFRs) couple to both the Ras/Raf/Erk pathway through Shc and Src, and PI3K lipid signaling through p85 (Valius and Kazlauskas, 1993). Cotransfection of the wild-type PDGFR and the c-fos reporter led to a 4-fold increase in c-fos–LacZ expression when cells were treated with PDGF (Figure 1C). PDGF stimulation was absent in cells transfected with the Y740F/Y751F receptor mutant, which does not couple to PI3K. In contrast, the Y771F mutant, which does not couple to Shc, showed activation of c-fos–LacZ expression comparable to the wild-type PDGFR-transfected cells. Taken together, these data suggest that the PI3K pathway is required for activation of c-fos transcription by NGF or PDGF. PI3K signaling is necessary for full activation of SRE-mediated gene expression by growth factors Because PI3K-regulated c-fos transcription targets the SRE, we explored the regulation of SRE-mediated transcription further by transfecting PC12 or 3T3-L1 cells with SRE–luciferase reporter constructs. NGF markedly activated PI3K as measured by Ser473 phosphorylation of the PI3K-regulated kinase, Akt (Figure 2B). Wortmannin and LY294002 inhibited NGF-stimulated SRE-mediated transcription in PC12 cells at concentrations consistent with specific inhibition of PI3K (Figure 2A). This inhibition was not due to non-specific effects on other SRE transactivating kinases since NGF-stimulated Erk, JNK and p38 phosphorylation were not attenuated by LY294002 treatment (Figure 2B). We also confirmed that inhibition of PI3K in PC12 cells and 3T3-L1 cells does not result in toxicity or downregulation of SRF expression (unpublished observations). As was the case for c-fos transcription, PC12 cells transfected with a PI3K-deficient PDGF receptor did not show PDGF-stimulated SRE-mediated transcription (Figure 2C). Cotransfection of DN p85 also attenuated mitogen-stimulated SRE-regulated transcription in PC12 and 3T3-L1 cells (Figure 2D). These data establish a role for PI3K in growth factor-stimulated SRE-mediated gene expression. Figure 2.NGF stimulation of SRE-mediated gene expression in PC12 is attenuated by inhibition of PI3K. (A) PI3K inhibitors decreased NGF-stimulated SRE-mediated gene expression. PC12 cells were transfected with a SRE–luciferase reporter plasmid, pretreated with the indicated concentrations of wortmannin or LY294002, treated with 100 ng/ml NGF and assayed for luciferase activity. NGF-stimulated, SRE-mediated transcription in the absence of inhibitor was set at 100%. (B) LY294002 inhibited PI3K signaling selectively. PC12 cells were serum starved for 18 h, pretreated with vehicle or 25 μM LY294002 (LY), treated with 100 ng/ml NGF for 10 min, lysed and analysed by western blot analysis for phosphorylated Erk, JNK, p38 and Akt. (C) PDGF receptor activation of SRE-mediated gene expression depended upon coupling to PI3K. PC12 cells were transfected with a SRE–luciferase reporter plasmid and PDGFβ receptor (PDGFR) (as described in Figure 1C). Cells were treated with 30 ng/ml PDGF and assayed for luciferase activity. (D) Dominant-negative PI3K inhibited SRE-mediated gene expression. PC12 and 3T3-L1 cells were transfected with a SRE–luciferase reporter plasmid along with a 3-fold excess of either empty vector or dominant-negative PI3K regulatory subunit (DN p85) expression vectors (125 ng/well). Cells were treated with 10% FBS, 100 ng/ml NGF or 50 ng/ml FGF, and assayed for luciferase activity. Where applicable, the data are means ± SD of triplicate assays normalized to EF1α promoter-driven LacZ expression. Download figure Download PowerPoint Expression of constitutively active PI3K (p110*) increased SRE-mediated transcription, indicating that PI3K activity is sufficient for SRE-dependent transcription (Figure 3A). Activated PI3K targeted SRF because mutation of the core SRF binding site (mSRF) blocked p110*-mediated transcription (Figure 3A). The ability of PI3K to couple to SRE-dependent transcription was not the result of crosstalk with MAPK cascades because p110* increased Akt phosphorylation but not phosphorylation of Erk, JNK or p38 MAPK (Figure 3B). Consistent with this, p110*-stimulated transcription was not inhibited by expression of dominant-negative (DN) MEK1 (MEK S222A), JBD (Jun kinase binding domain of JIP) or DN Rho N19 (unpublished observations). This led us to examine whether downstream targets of PI3K regulate SRE-mediated gene expression. Through the use of selective inhibitors or dominant negatives we ruled out a major role for atypical PKCs, conventional PKCs, p70 S6 kinase and Akt in mitogen-induced SRE-mediated transcription (unpublished observations). PI3K signaling regulates activation and nuclear translocation of serum- and glucocorticoid-inducible kinase (SGK) (Kobayashi and Cohen, 1999; Park et al., 1999), making it an attractive candidate for a downstream SRF effector. Expression of constitutively active SGK, but not Akt, markedly increased mEts SRE–luciferase reporter gene expression (Figure 3C). Both mutants were active because expression of activated Akt and SGK in PC12 cells increased GSK3 phosphorylation (Figure 3D). This suggests that SGK is a major PI3K-regulated effector of SRF-regulated transcription. Collectively these data indicate that PI3K is both necessary and sufficient for growth factor-stimulated SRE-mediated gene expression in PC12 cells and 3T3-L1 cells. Figure 3.Activation of PI3K and SGK induces SRE-mediated transcription. (A) Constitutively active PI3K increased SRE-mediated gene expression. PC12 cells were transfected with either a wild-type SRE luciferase reporter (SRE–luc) or a mutant SRE reporter (mSRF SRE–luc) that cannot bind SRF. Cells were cotransfected with increasing amounts of a constitutively active PI3K (p110*) expression vector. The data are means ± SEM of triplicate assays normalized to EF1α-driven LacZ expression. (B) Expression of p110* selectively activated PI3K-dependent signaling. PC12 cells were transfected with control or p110* expression vectors. Cells were treated with 100 ng/ml NGF for 10 min. Cells transfected with p110* were lysed 36 h post-transfection. Lysates were western blotted for phosphorylated Akt, Erk, JNK or p38 MAPK. (C) Expression of activated SGK increased SRF-dependent gene expression. PC12 cells were transfected with an SRF-regulated luciferase reporter that contains a mutation in the TCF binding Ets site (mEts SRE–luc) and a 3-fold excess of empty, constitutively active Akt (CA Akt) or constitutively active SGK (CA SGK) expression vectors. The data are means ± SEM of triplicate assays normalized to SV40-driven SEAP expression. (D) Expression of activated SGK and activated Akt increased GSK3 phosphorylation. PC12 cells were transfected with EF1α–LacZ and vector, activated Akt (CA Akt) or activated SGK (CA SGK), and extracts were blotted for phospho-GSK3. The extracts were then blotted for LacZ to control for transfection efficiency. Download figure Download PowerPoint Since SRE-mediated gene expression can be activated by several MAPK cascades, including the ERK, p38 MAPK and JNK pathways (for a review see Treisman, 1995), we determined to what extent these pathways contribute to mitogen-stimulated transcription. We inhibited ERK, JNK and p38 MAPK signaling by expressing dominant-negative inhibitors or by treating cells with specific inhibitors (Table I). Inhibition of MAPK signaling either had no effect or only modestly inhibited SRE-mediated transcription (Table I). In every case, positive controls were performed to confirm that each inhibitor or dominant negative potently blocked their respective targets. For example, DN MEK and PD98059 blocked Gal4–Elk1-mediated transcription 87 and 98%, respectively (Figure 4G). These observations indicate that the PI3K pathway plays a major role in mitogen-stimulated SRE-mediated transcription. Figure 4.PI3K and mitogens regulate SRE-mediated gene expression by transactivating SRF but not TCF. (A) Mutations in the DNA binding sequences for SRF, but not Ets, abolished NGF-stimulated SRE-mediated transcription. SRE reporter plasmids were constructed containing mutations in the SRF (mSRF SRE–luc) or Ets (mEts SRE–luc) binding sites (see Materials and methods for sequences) and transfected into PC12 cells. Cells were treated with 100 ng/ml NGF, then assayed for luciferase activity. (B) Ets DNA binding mutations reduced growth factor-induced SRE-mediated gene expression in HeLa cells. Cells were transfected as in (A) with the wild-type and mEts SRE–luciferase reporter plasmids, treated with 50 ng/ml EGF and assayed. (C) SRF was required for NGF-stimulated SRE-mediated transcription. PC12 cells were transfected with equal amounts (75 ng/well) of a SRE–luciferase reporter plasmid and expression vectors for wild-type SRF (wt SRF), a dominant-negative SRF with a C-terminal truncation (SRF Δ) or a dominant-negative SRF deficient for DNA binding (SRFpm1). Cells were treated with 100 ng/ml NGF and assayed for luciferase activity. (D) Mitogens transactivated Gal4–SRF. PC12 cells were transfected with a Gal4–luciferase reporter plasmid and a 2-fold excess (67 ng/well) of either Gal4 DNA binding domain (Gal4) or Gal4-SRF expression vectors. Cells were stimulated with 10% fetal bovine serum (serum) or 100 ng/ml NGF, and assayed for luciferase activity. (E) Blocking PI3K, but not MEK or JNK, inhibited NGF stimulation of Gal4–SRF-mediated transcription. PC12 cells were transfected with a Gal4–luciferase reporter, Gal4–SRF and a 4-fold excess of DN p85 (200 ng/well), a 3-fold excess of DN MEK (133 ng/well) or JBD (133 ng/well) expression constructs. Cells were stimulated with 100 ng/ml NGF and assayed for luciferase activity. (F) MEK inhibitors, but not PI3K inhibitors, blocked phosphorylation of Elk1 at Ser383. PC12 cells transfected with Elk1 (pCMV Elk1) DNA, were pretreated with dimethylsulfoxide (DMSO), 10 μM PD98059 or 25 μM LY294002, then blotted for Elk1, Akt and Erk phosphorylation following treatment with 100 ng/ml NGF for 10 min. An SV40-LacZ expression vector was cotransfected and lysates were blotted for β-galactosidase as a control for transfection efficiency. (G) Inhibition of MEK, but not PI3K, attenuated transactivation of Gal4–Elk1. PC12 cells were transfected with a Gal4–luciferase reporter construct, a 2-fold excess of Gal4–Elk1 expression vector (89 ng/well) and a 3-fold excess of control or DN MEK expression plasmids (167 ng/well). Cells were pretreated with 25 μM LY294002, then treated with 100 ng/ml NGF and assayed for luciferase activity. Where applicable, the data are means ± SD of triplicate assays normalized to EF1α promoter-driven LacZ expression. Download figure Download PowerPoint Table 1. Effects of dominant negative and chemical inhibitors on growth factor stimulation of SRE-mediated gene expression Dominant negative/inhibitor % Inhibition of stimulationa by Serum PC12 cells NGF PC12 cells Serum 3T3-L1 cells MEK1b 12.4 ± 0.6 3.8 ± 1.0 0 ± 0.6 PD98059c 0 ± 0.2 10.5 ± 1.0 8.4 ± 0.3 JBDb 0 ± 4.6 10.9 ± 1.0 0 ± 7.0 SB203580c 5.3 ± 1.0 15.1 ± 1.0 5.6 ± 1.7 LY294002c 78.2 ± 27.4 82.7 ± 2.4 49.7 ± 7.8 a PC12 and 3T3-L1 cells were transfected with a SRE–luciferase reporter, serum starved for 18 h, then treated with either 10% fetal bovine serum (Serum) or 100 ng/ml NGF for 5 h. b Dominant-negative MEK1 and JBD were cotransfected at a 3-fold excess (133 ng/well) of dominant negative to reporter constructs. Constructs are referenced in Materials and methods. c Cells were pretreated with 25 μM PD98059, 5 μM SB203580, 25 μM LY294002 for 1 h. Values are the mean of triplicate assays ± SD normalized to EF1α-driven LacZ expression. Growth factors stimulate SRE-mediated transcription independently of Rho signaling The Rho family of small G-proteins regulate c-fos expression and SRF-dependent transcription via LIMK-1-regulated actin treadmilling (Sotiropoulos et al., 1999). To determine whether Rho family G-proteins contribute to PI3K-regulated gene expression, PC12 cells were transfected with a SRE–luciferase reporter construct and inhibitors of Ras (Ras N17), Rho (C3 exoenzyme, Rho GDI and Rho N19), Rac (Rac N17) or Cdc42 (Cdc42 N17). Surprisingly, expression of these dominant negatives was either without effect or only slightly decreased mitogen-induced SRE-regulated transcription (Table II). Interestingly, the modest inhibition of SRE-mediated transcription by these dominant negatives (5–13%; Table II) was far less than that seen following inhibition of PI3K (57–82%; Figure 2D and Table I), indicating that in PC12 cells the Rho transcriptional pathway is dispensable for mitogen-stimulated SRE-regulated transcription. The potent efficacy of each inhibitory construct was confirmed in PC12 and NIH 3T3 cells (unpublished observations). Therefore, we propose a novel PI3K-dependent pathway for the activation of SRF-mediated transcription that does not involve Rho family G-proteins. Table 2. Inhibition of Rho family small G proteins does not affect mitogen stimulation of SRE-mediated gene expression Expression constructsb % Inhibition of stimulationa by Serum NGF Ras N17 0 ± 1.0 0 ± 6.1 C3 12.5 ± 0.7 13.0 ± 0.5 Rho GDI 0 ± 0.1 4.5 ± 0.1 Rho N19 0 ± 1.1 7.7 ± 0.9 Rac N17 0 ± 2.2 10.6 ± 1.2 Cdc42 N17 0 ± 1.5 10.4 ± 0.4 a PC12 cells were transfected with a SRE–luciferase reporter plasmid, serum starved for 18 h, then treated with either 10% FBS (Serum) or 100 ng/ml NGF for 5 h, then assayed for luciferase activity. b A 3-fold excess (133 ng/well) of either control vector, C3 botulinum toxin expression vector, a Rho guanine nucleotide dissociation inhibitor (Rho GDI) dominant-negative Rho (Rho N19), Cdc42 (Cdc42 N17) or Rac (Rac N17) to reporter was cotransfected. Values are the mean of triplicate assays ± SD. Values are normalized for transfection efficiency by cotransfection of an EF1α–LacZ expression vector and measurement of β-galactosidase activity from cell lysates. PI3K activation of SRE-mediated gene expression occurs via SRF Several transcription factors bind to the c-fos SRE and regulate transcription. To determine the target of PI3K-dependent signaling, we generated mutant reporter constructs that are deficient for binding of SRF (mSRF) or TCF (mEts) to the SRE. PC12 cells transfected with the mSRF luciferase reporter construct did not show NGF-induced transcription (Figure 4A). Disruption of the TCF site 5′ of the CArG had little effect on NGF stimulation of the SRE. Although the ETS TCF pathway plays an important role in SRE-regulated transcription in fibroblasts (Hill et al., 1993), in other cell types different pathways predominate (Johansen and Prywes, 1994; Hill and Treisman, 1995). For example, disruption of the TCF binding site reduced EGF-induced SRE-mediated transcription (Figure 4B). Nevertheless, significant stimulation remained, indicating a TCF-independent target for polypeptide growth factors at the SRE. We also confirmed that SRE-mediated gene expression required SRF via the use of two specific SRF dominant negatives. One is a mutant with a deleted C-terminal activation domain, which forms transcriptionally inactive heterodimers with wild-type SRF (SRF Δ). The other mutant is a DNA binding domain mutant that forms heterodimers with wild-type SRF, reducing the pool of DNA binding competent SRF (SRFpm1). Both dominant-negative SRF mutants inhibited NGF-stimulated SRE-mediated transcription (Figure 4C), but not forskolin-stimulated CRE-mediated transcription (unpublished observations). To confirm that SRF is transactivated by PI3K signaling, we examined the ability of fusions of full-length SRF to the yeast Gal4 DNA binding domain (Gal4–SRF) to drive Gal4–luciferase transcription. Serum and NGF transactivated Gal4–SRF (Figure 4D). Transactivation of SRF required PI3K, as LY294002 and DN p85 both inhibited NGF stimulation of transcription (Figure 4E). Similarly, expression of DN MEK and JBD had no effect on activation of Gal4–SRF by NGF (Figure 4E). These data argue that SRF or a novel accessory protein is a direct target of PI3K-dependent signaling. Because the ETS family TCFs, Elk1 and Sap1, play a crucial role in mitogen-stimulated SRE-dependent transcription (Treisman, 1995), we examined whether they are also a target of PI3K signaling. Treatment of PC12 cells with LY294002 did not reduce NGF-stimulated phosphorylation of Ser383 of Elk1, a residue critical for Elk1 transactivation (Figure 4E). While LY294002 treatment did not affect transactivation of Gal4–Elk1 (Figure 4G), it markedly attenuated the transactivation of Gal4–SRF. Conversely, PD98059, which had no effect on NGF stimulation of SRE-mediated transcription (Table I), strongly inhibited Elk1 phosphorylation (Figure 4F). Similarly, expression of DN MEK attenuated transactivation of Gal4–Elk1 (Figure 4G) but was without effect on Gal4–SRF. These data strongly suggest that SRF or a novel accessory factor is the primary target of a PI3K-dependent and polypeptide growth factor-mediated transcriptional cascade. PI3K and SRF are required for mitogen-induced cell cycle progression Several lines of evidence suggest that PI3K plays a critical role in cellular proliferation. Although several studies report that PI3K may promote cell cycle progression by activating p70 S6 kinase (Klippel et al., 1998), other studies suggest an unknown mechanism (Gille and Downward, 1999; Takuwa et al., 1999). Thus, the mechanism(s) by which PI3K signaling regulates cell cycle progression is not clear. Because PI3K can potently activate SRF-dependent gene expression, we explored whether SRF is a potential target of PI3K during cell cycle progression. Cotransfection of regulatory constructs with a chloramphenicol acetyl transferase (CAT) expression vector enabled us to identify transfected cells and assess whether DNA synthesis occurred, using immunocytochemistry (ICC) for CAT detection and BrdU incorporation, respectively. Treatment of quiescent 3T3-L1 cells with serum or fibroblast growth factor (FGF) resulted in a marked increase in DNA synthesis measured by ICC for BrdU incorporation (Figure 5A and B). Expression of DN p85 attenuated serum-induced BrdU labelling, confirming that PI3K signaling is critical for the G1–S transition (Figure 5A and B). If SRF is an important target of PI3K during the G1–S transition, then inhibition of SRF-dependent transcription should also attenuate entry into the cell cycle. Expression of SRF Δ or SRFpm1 attenuated serum- or FGF-induced cell cycle progression relative to vector control (Figure 5A and B). FACS analysis confirmed that expression of SRF Δ and DN p85 inhibits serum- stimulated cell cycle progression in G0/G1 (Figure 5C). Figure 5.SRF-regulated transcription and PI3K activity are obligatory for mitogen-stimulated cell cycle progression. (A) Inhibition of SRF-dependent gene expression or PI3K activity blocked mitogen-stimulated cell cycle entry. Representative images depict BrdU stai
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