Title: Identification of Two Sp1 Phosphorylation Sites for p42/p44 Mitogen-activated Protein Kinases
Abstract: Sp1 regulates activation of many genes implicated in tumor growth and cell cycle progression. We have previously demonstrated its implication in the up-regulation of vascular endothelial growth factor (VEGF) gene transcription following growth factor stimulation of quiescent cells, a situation where p42/p44 mitogen-activate protein kinase (MAPK) activity is dramatically increased. Here we show that p42/p44 MAPK directly phosphorylates Sp1 on threonines 453 and 739 both in vitro and in vivo. Mutation of these sites to alanines decreases by half the MAPK-dependent transcriptional activity of Sp1, in the context of the VEGF promoter, in SL2 Drosophila cells devoid of the endogenous Sp1 protein. Moreover, inducible overexpression of the (T453A,T739A) Sp1 double mutant compromises MAPK-driven VEGF mRNA transcription in fibroblasts. These results highlight Sp1 as a key molecular link between elevated activation of the Ras ≫ p42/p44MAPK signaling pathway and increased VEGF expression, two major steps deregulated in tumor cells. Sp1 regulates activation of many genes implicated in tumor growth and cell cycle progression. We have previously demonstrated its implication in the up-regulation of vascular endothelial growth factor (VEGF) gene transcription following growth factor stimulation of quiescent cells, a situation where p42/p44 mitogen-activate protein kinase (MAPK) activity is dramatically increased. Here we show that p42/p44 MAPK directly phosphorylates Sp1 on threonines 453 and 739 both in vitro and in vivo. Mutation of these sites to alanines decreases by half the MAPK-dependent transcriptional activity of Sp1, in the context of the VEGF promoter, in SL2 Drosophila cells devoid of the endogenous Sp1 protein. Moreover, inducible overexpression of the (T453A,T739A) Sp1 double mutant compromises MAPK-driven VEGF mRNA transcription in fibroblasts. These results highlight Sp1 as a key molecular link between elevated activation of the Ras ≫ p42/p44MAPK signaling pathway and increased VEGF expression, two major steps deregulated in tumor cells. Normal and pathological angiogenesis depends on the secretion of growth factors needed for proliferation and survival of endothelial cells. Among these factors the vascular endothelial growth factor (VEGF), 1The abbreviations used are: VEGFvascular endothelial growth factorMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseGSTglutathione S-transferaseCMVcytomegalovirusMEKMAPK/ERK kinasePBSphosphate-buffered salineEMSAelectrophoretic mobility shift assayBSAbovine serum albuminSAPKstress-activated protein kinaseJNKc-Jun NH2-terminal kinaseWTwild typeHAhemagglutinin which is overexpressed by a wide variety of human tumors (1Berse B. 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Pagès G. J. Biol. Chem. 1998; 273: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). However, the phosphatidylinositol 3-kinase pathway via activation of protein kinase C ζ is also highly important in human fibrosarcoma and renal cell carcinoma for driving VEGF transcription (23Pal S. Datta K. Khosravi-Far R. Mukhopadhyay D. J. Biol. Chem. 2000; 276: 2395-2403Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 24Rak J. Mitsuhashi Y. Sheehan C. Tamir A. Vioria-Petit A. Filmus J. Mansour S.J. Ahn N.G. Kerbel R.S. Cancer Res. 2000; 60: 490-498PubMed Google Scholar). In both cases, the main transcription factor implicated in the regulation of VEGF transcription following Ras activation is Sp1 (8Pal S. Claffey K.P. Cohen H.T. Mukhopadhyay D. J. Biol. Chem. 1998; 273: 26277-26280Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 22Milanini J. Vinals F. Pouysségur J. Pagès G. J. Biol. 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This region containing two Sp1 sites actually binds the transcription factor (22Milanini J. Vinals F. Pouysségur J. Pagès G. J. Biol. Chem. 1998; 273: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Another study showed that recombinant active p42 MAPK enhances DNA binding capacity of Sp1 in vitro (56Merchant J.L., Du, M. Todisco A. Biochem. Biophys. Res. Commun. 1999; 254: 454-461Crossref PubMed Scopus (179) Google Scholar). These observations prompted us to test the hypothesis that Sp1 is a link between the MAPK pathway and VEGF expression. First we observed that MAPK stimulation rapidly enhanced DNA binding of Sp1 and Sp3 to the VEGF promoter. By using in vitro kinase assays as well as antibodies directed against phosphopeptides we identified two major sites targeted by p42/p44 MAPK on Sp1. Both sites are indispensable for Sp1 activity following activation of the p42/p44 MAPK pathway. Thereafter, we demonstrated that phosphorylation of Sp1 by p42/p44 MAPK is a crucial event for the regulation of at least VEGF, one of the key genes implicated in neovascularization. Restriction and DNA modifying enzymes were obtained from New England BioLabs or from Eurogentec (Liège, Belgium). [α-32P]dCTP and [α-32P]dATP were from ICN. Synthetic oligonucleotides were from Eurogentec. Recombinant human proteins Sp1 and AP-2 were purchased from Promega. Recombinant active p42 MAPK/ERK2 was purchased from New England BioLabs. Anti-Myc antibody (9E10) was from Roche Molecular Biochemicals. Anti-Sp1 (PEP-2) was from Santa Cruz Biotechnology. ΔRaf:ER cells are a derivative of CCL39 fibroblasts that stably expressed a fusion protein comprised of the catalytic domain of Raf-1 and the hormone binding domain of the estrogen receptor (19Samuels M.L. Weber J.M. McMahon M. Mol. Biol. Cell. 1993; 13: 6241-6252Crossref Scopus (323) Google Scholar, 20Samuels M.L. McMahon M. Mol. Biol. Cell. 1994; 14: 7855-7866Crossref Google Scholar, 21Lenormand P. McMahon M. Pouyssegur J. J. Biol. Chem. 1996; 271: 15762-15768Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). These cells were cultivated in Dulbecco's modified Eagle's medium (Invitrogen) without phenol red containing 7.5% fetal calf serum, penicillin (50 units/ml), streptomycin sulfate (50 μg/ml), and G418 (400 μg/ml). Growth-arrested cells were obtained by total deprivation of serum for 48 h. Cells expressing a tetracycline-inducible vector were cultivated in the same medium supplemented with blasticidin (7.5 μg/ml, selection of the tetracycline repressor) and zeocin (500 μg/ml, selection of the gene of interest). Induction of the transgene was obtained by stimulating the cells for 24–48 h with 1 μg/ml tetracycline. The Drosophila melanogaster Schneider (SL2) cells were grown in DES medium (Invitrogen) withl-glutamine supplemented with 10% heat-inactivated fetal calf serum. ΔRaf:ER cells (106 cells/10-cm diameter dish) were transfected by CaPO4 precipitation technique with 15 μg of the different pcDNA4/TO vectors. SL2 cells were transiently transfected as described above (see luciferase assays). All the plasmids coding for the different subdomains of Sp1 fused to GST were a generous gift of Dr. J. Horowitz excepted the GST-D, which was obtained by PCR using the following oligonucleotides on the GST-Zn matrix: forward, 5′-CGGGATCCCGGCACTGCCACTCCTTCAGCC-3′; reverse, 5′-GGAATTCCTAGTTGGCAAGACGGGCAATGC-3′ (57Murata Y. Kim H.G. Rogers K.T. Udvadia A.J. Horowitz J.M. J. Biol. Chem. 1994; 269: 20674-20681Abstract Full Text PDF PubMed Google Scholar). The full-length Sp1 cDNA excised from the pGEX vector was introduced in the pCMVTag vector within EcoRI/BamHI sites. We then excised a NotI/XhoI fragment of this vector and introduced it in the pcDNA4/TO vector (Invitrogen). The different point mutations of the MAPK consensus phosphorylation sites were obtained using a QuikChange site-directed mutagenesis kit supplied by Stratagene. The vector containing the VEGF promoter fused to the luciferase reporter gene has already been described (22Milanini J. Vinals F. Pouysségur J. Pagès G. J. Biol. Chem. 1998; 273: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The MEKSD/SE-MAPK construct corresponding to a fusion of the MEKSD/SE cDNA with the ERK2 cDNA (kindly provided by Dr. Y. Miyata (58Miyata Y. Adachi S. Mizuno H. Nishida E. Biochim. Biophys. Acta. 1999; 1451: 334-342Crossref PubMed Scopus (7) Google Scholar)), was subcloned in the XhoI site of the pCMVTag3B vector (Stratagene). Cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed in the “RNA Insta-Pure” buffer from Eurogentec. The supernatant was cleared by centrifugation, ethanol-precipitated, and resuspended in sterile water. Ten micrograms of RNA was used for Northern analysis and analyzed as previously described (22Milanini J. Vinals F. Pouysségur J. Pagès G. J. Biol. Chem. 1998; 273: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 59Pagès G. Berra E. Milanini J. Levy A.P. Pouysségur J. J. Biol. Chem. 2000; 275: 26484-26491Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). SL2 Drosophila cells in 12-well dishes (105/well) were transiently transfected using the calcium phosphate technique. 3.5 μg of reporter plasmid (−88/+54 of the VEGF promoter in pGL2 basic vector) was co-transfected with 0.5 μg of Myc-Sp1-HA (wt or mutants), in the absence or presence of 1 μg of MEKSD/SE-MAPK. 1.5 μg of pPAC plasmid coding for the LacZ gene under control of the Drosophila actin promoter was also added as a control of transfection efficiency. Salmon sperm DNA was added to reach a final amount of 13 μg. Sixteen hours after transfection, cells were washed once in DES medium and incubated in DES medium supplemented with 10% heat-inactivated fetal bovine serum. Four days after transfection, cells were washed in PBS, and luciferase assays were performed as previously described (22Milanini J. Vinals F. Pouysségur J. Pagès G. J. Biol. Chem. 1998; 273: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar) Confluent ΔRaf:ER cell cultures were serum-deprived overnight prior to stimulation with 1 μmestradiol for 15 min. Nuclear extracts, electromobility shift assays (EMSA), and supershift assays were performed, as previously described (22Milanini J. Vinals F. Pouysségur J. Pagès G. J. Biol. Chem. 1998; 273: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The probe used in these experiments was synthesized to span the region of the human VEGF promoter comprised between the −88 and −66 bp: 5′-TTTCCGGGGCGGGCCGGGGGCGGGG TAT-3′ (random sequences added to the wild type sequence are shown in italic letters). Anti-phosphopeptide sera were generated by Neosystem (Strasbourg, France) by injecting two rabbits each with the following phosphopeptides. Phosphopeptide 1 (Phospho Thr739): NH2-KRRSEGSTA-(PO3H2)-T PSAlI-COOH coupled to KLH. Phosphopeptide2 (Phospho Thr453): NH2-KSGPIIIR-(PO3H2)-T PTVGPNG-COOH (where the boldface “T” represents the threonines targeted by MAPK), coupled to ovalbumin (GenBankTM accession numbers: AB039286 and J03133). Sera were affinity-purified by passing them first over an EAH-Sepharose 4B column (Amersham Biosciences, Inc.) to which the unphosphorylated peptide was coupled and the flow-through was collected. The non-retained fraction was then passed over a column to which the phosphorylated peptide was bound. Specific IgG were then eluted with 100 mm glycine (pH 2.8) and neutralized in Tris 3 m, pH 11. ΔRaf:ER cells were plated on glass coverslips at a density of 105 cells/35-mm dish. The cells were rendered quiescent by incubation in serum-free medium for 24 h and stimulated or not with estradiol 1 μm. Cells were then fixed with 10% paraformaldehyde at 37 °C, followed by methanol permeabilization for 15 min at −20 °C. Coverslips were washed with PBS, and the nonspecific sites were blocked by incubation with PBS containing 2% bovine serum albumin (BSA) and 0.2% gelatin. Coverslips were incubated with the first antibody diluted in PBS/BSA/gelatin (anti-phospho-Thr453, 1/120; anti-Myc, 1/1000) for 1 h, then washed five time with PBS. Prior to the incubation with the second antibody (biotin-conjugated goat anti-rabbit, 1/1000 and fluorescein isothiocyanate-conjugated goat anti-mouse, 1/100), 4′,6-diamidine dihydrochloride (Roche) was added at a final concentration of 0.2 μg/ml during the last 15 min of incubation to enumerate cells. After extensive washes in PBS and in distilled water, coverslips were mounted in Citifluor and examined under epifluorescence illumination. They were performed in kinase buffer (20 mm Tris, pH 7.5, 10 mmpara-nitrophenyl phosphate, 10 mmMgCl2, 2 mm dithiothreitol) with ∼5 μg of the different GST/Sp1 fusion proteins or equimolar amounts of Sp1 (0.6 μg), AP-2 (0.3 μg), BSA (0.4 μg), myelin basic protein (0.12 μg), GST-ATF2 (0.4 μg), GST-Elk1 (0.3 μg), and GST-Jun (0.4 μg) as substrates and 5 μCi of 50 μm[γ-32P]ATP for 15 min at 30 °C. The reaction was stopped by addition of Laemmli sample buffer and resolved on SDS-PAGE. ΔRaf:ER cells or ΔRaf:ER cells stably transfected with the tetracycline inducible vectors were serum-deprived for 48 h. After estradiol or serum stimulation, cells were washed with ice-cold PBS and immediately lysed in Laemmli sample buffer. Eighty micrograms of protein was resolved by SDS-PAGE on 7.5% gels and transferred onto a polyvinylidene difluoride membrane (Immobilon). The membranes were incubated with purified anti-phospho-Thr739 antibody (1/1000) or anti-phospho-MAPK (1/5000) or anti-total Sp1 (PEP2 Santa Cruz Biotechnology, 1/2000). The protein was labeled with an anti-rabbit horseradish peroxidase-conjugated secondary antibody and developed using an ECL system (Amersham Biosciences, Inc.). We have previously shown that long term activation of the p42/p44 MAPK pathway results in increased VEGF transcription. This effect was found to be directly dependent on the recruitment of Sp1 and AP-2 transcription factors to a GC-rich region located on the proximal region of the VEGF promoter (−88/−66) (22Milanini J. Vinals F. Pouysségur J. Pagès G. J. Biol. Chem. 1998; 273: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). To determine whether this effect of p42/p44MAPK on the DNA binding activity of these transcription factors is direct, we performed EMSA experiments with nuclear extracts of cells expressing an estradiol-inducible Raf-1 (ΔRaf-1:ER) (19Samuels M.L. Weber J.M. McMahon M. Mol. Biol. Cell. 1993; 13: 6241-6252Crossref Scopus (323) Google Scholar, 20Samuels M.L. McMahon M. Mol. Biol. Cell. 1994; 14: 7855-7866Crossref Google Scholar). In these cells, p42/p44 MAPK activity is rapidly and exclusively activated by estradiol (21Lenormand P. McMahon M. Pouyssegur J. J. Biol. Chem. 1996; 271: 15762-15768Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). ΔRaf-1:ER cells were serum-deprived for 48 h and then stimulated by the addition of estradiol. When incubated with nuclear extracts of untreated ΔRaf-1:ER cells, a basal DNA binding activity on a double-stranded probe encompassing the −88/−66-bp region of the human VEGF promoter was detected (Fig. 1A, lane 1). In nuclear extracts of cells stimulated with estradiol for 15 min (Fig. 1A, lane 3) or 3 h (Fig. 1A, lane 5), an increase DNA binding activity was observed. Both basal and the stimulated complexes, in extracts of estradiol-stimulated cells for 15 min (Fig. 1A, lane 4) or 3 h (Fig. 1A, lane 6), were almost entirely disrupted in the presence of an excess of unlabeled double-stranded Sp1 consensus oligonucleotide, demonstrating that these complexes contain Sp1 or Sp1-related proteins. However, when extracts of cells stimulated with estradiol for 3 h (Fig. 1A, lane 6) were used, one part of complex B resists competition with unlabeled Sp1 consensus oligonucleotide. One part of this complex can be attributed to the presence of the AP-2 transcription factor, which is recruited to the VEGF promoter after long term p42/p44 MAPK stimulation as previously described (22Milanini J. Vinals F. Pouysségur J. Pagès G. J. Biol. Chem. 1998; 273: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). To demonstrate the presence of Sp1 in the different complexes observed with extracts of estradiol-stimulated ΔRaf-1:ER cells for 15 min, we performed supershift experiments with anti-Sp1 antibody. Our results clearly demonstrate that Sp1 (present at least in complexes B1 and B2) is recruited to the VEGF promoter following a short term stimulation of p42/p44 MAPK by estradiol (Fig. 1B). Supershift experiments with anti-Sp3 antibody also show that Sp3 is recruited to the VEGF promoter following a short term stimulation with estradiol and is contained within complex B3 and c (data not shown). Band shift as well as supershift experiments were also performed with nuclear extracts of estradiol-stimulated cells in the presence of cycloheximide, an inhibitor of protein synthesis. The same results were also obtained in such conditions (data not shown). Because this effect occurred rapidly and in conditions where protein neo-synthesis is blocked, we postulated that it could be accounted for by direct phosphorylation of the transcription factors. We have then investigated whether Sp1 could be directly phosphorylated by p42/p44 MAPK. Thus, we performed in vitro kinase assays using equivalent molar amounts of affinity-purified Sp1, AP-2, bovine serum albumin (BSA), GST-ATF2, GST-Jun, GST-Elk1, and myelin basic protein. GST-ATF2, a specific substrate for the SAPK/c-Jun NH2-terminal kinase and p38/HOG MAPK (60Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1339) Google Scholar, 61Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (476) Google Scholar), GST-Jun, a specific substrate for the SAPK/c-Jun NH2-terminal kinase (62Derijard B. Hibi M. Wu I.H. Barrett T., Su, B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar) and BSA can be considered as negative controls. GST-Elk1 (63Hipskind R.A. Buscher D. Nordheim A. Baccarini M. Genes Dev. 1994; 8: 1803-1816Crossref PubMed Scopus (108) Google Scholar, 64Hipskind R.A. Baccarini M. Nordheim A. Mol. Cell. Biol. 1994; 14: 6219-6231Crossref PubMed Scopus (137) Google Scholar) and myelin basic protein (65Meloche S. Pagès G. Pouysségur J. Mol. Biol. Cell. 1992; 3: 63-71Crossref PubMed Scopus (131) Google Scholar), commonly used substrates for p42/p44 MAPK, are considered as positive controls. We observed that recombinant active p42 MAPK strongly phosphorylates Sp1 as well as GST-E