Identification of Two Sp1 Phosphorylation Sites for p42/p44 Mitogen-activated Protein Kinases

• DOI: 10.1074/jbc.m201753200
• Publication Date: 2002-07-26T15:16:37Z
• Authors: Julie Milanini-Mo...; Jacques Pouysségur; Gilles Pagès
• Keywords: Sp1 transcription factor

Sp1 regulates activation of many genes implicated in tumor growth and cell cycle progression. We have previously demonstrated its implication the up-regulation vascular endothelial factor (VEGF) gene transcription following stimulation quiescent cells, a situation where p42/p44 mitogen-activate protein kinase (MAPK) activity is dramatically increased. Here we show that MAPK directly phosphorylates on threonines 453 739 both vitro vivo. Mutation these sites to alanines decreases by half MAPK-dependent transcriptional Sp1, context VEGF promoter, SL2 Drosophila cells devoid endogenous protein. Moreover, inducible overexpression (T453A,T739A) double mutant compromises MAPK-driven mRNA fibroblasts. These results highlight as key molecular link between elevated Ras ≫ p42/p44MAPK signaling pathway increased expression, two major steps deregulated cells. 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Tsuoro Oka Yoshida Shimada Ari Wada Fujimoto Kohno 813-822Crossref (614) Concomitantly, contributes neovascularization (55Risau 386: 671-674Crossref (4867) previous short region MAPK. This containing actually binds Another showed recombinant active p42 enhances DNA capacity (56Merchant J.L., Todisco 254: 454-461Crossref (179) observations prompted us test hypothesis First rapidly enhanced Sp3 promoter. By using assays well antibodies directed against phosphopeptides Sp1. Both indispensable pathway. Thereafter, event least VEGF, Restriction modifying enzymes were obtained New England BioLabs or Eurogentec (Liège, Belgium). [α-32P]dCTP [α-32P]dATP ICN. Synthetic oligonucleotides Eurogentec. Recombinant AP-2 purchased Promega. MAPK/ERK2 BioLabs. Anti-Myc antibody (9E10) Roche Molecular Biochemicals. Anti-Sp1 (PEP-2) Santa Cruz Biotechnology. ΔRaf:ER derivative stably expressed fusion comprised catalytic domain Raf-1 hormone estrogen receptor cultivated Dulbecco's modified Eagle's medium (Invitrogen) without phenol red 7.5% fetal calf serum, penicillin (50 units/ml), streptomycin sulfate μg/ml), G418 (400 μg/ml). Growth-arrested total deprivation 48 h. Cells expressing tetracycline-inducible vector same supplemented with blasticidin (7.5 μg/ml, selection tetracycline repressor) zeocin (500 interest). Induction transgene stimulating 24–48 h 1 μg/ml tetracycline. melanogaster Schneider (SL2) grown DES withl-glutamine 10% heat-inactivated serum. (106 cells/10-cm diameter dish) transfected CaPO4 precipitation technique 15 μg different pcDNA4/TO vectors. transiently (see luciferase assays). All plasmids coding subdomains fused GST generous gift Dr. Horowitz excepted GST-D, PCR GST-Zn matrix: forward, 5′-CGGGATCCCGGCACTGCCACTCCTTCAGCC-3′; reverse, 5′-GGAATTCCTAGTTGGCAAGACGGGCAATGC-3′ (57Murata H.G. Rogers K.T. Udvadia 269: 20674-20681Abstract full-length cDNA excised pGEX introduced pCMVTag within EcoRI/BamHI sites. then NotI/XhoI fragment (Invitrogen). point mutations consensus QuikChange site-directed mutagenesis kit supplied Stratagene. promoter reporter already MEKSD/SE-MAPK construct corresponding MEKSD/SE ERK2 (kindly provided Miyata (58Miyata Adachi Mizuno Nishida Biochim. Acta. 1451: 334-342Crossref (7) Scholar)), subcloned XhoI site pCMVTag3B (Stratagene). washed ice-cold (PBS) lysed “RNA Insta-Pure” buffer supernatant cleared centrifugation, ethanol-precipitated, resuspended sterile water. Ten micrograms RNA Northern analysis analyzed 59Pagès Berra Milanini 275: 26484-26491Abstract 12-well dishes (105/well) calcium phosphate technique. 3.5 plasmid (−88/+54 pGL2 basic vector) co-transfected 0.5 Myc-Sp1-HA (wt mutants), absence presence MEKSD/SE-MAPK. 1.5 pPAC LacZ under actin added transfection efficiency. Salmon sperm reach final amount 13 μg. Sixteen hours after transfection, incubated Four days PBS, performed Confluent cultures serum-deprived overnight prior μmestradiol min. Nuclear extracts, electromobility (EMSA), supershift performed, probe experiments synthesized span −88 −66 bp: 5′-TTTCCGGGGCGGGCCGGGGGCGGGG TAT-3′ (random sequences sequence italic letters). Anti-phosphopeptide sera generated Neosystem (Strasbourg, France) injecting rabbits each phosphopeptides. Phosphopeptide (Phospho Thr739): NH2-KRRSEGSTA-(PO3H2)-T PSAlI-COOH coupled KLH. Phosphopeptide2 Thr453): NH2-KSGPIIIR-(PO3H2)-T PTVGPNG-COOH (where boldface “T” represents MAPK), ovalbumin (GenBankTM accession numbers: AB039286 J03133). Sera affinity-purified passing them over EAH-Sepharose 4B column (Amersham Biosciences, Inc.) unphosphorylated peptide flow-through collected. non-retained fraction passed bound. Specific IgG eluted 100 mm glycine (pH 2.8) neutralized Tris 3 m, pH 11. plated glass coverslips density 105 cells/35-mm dish. rendered incubation serum-free 24 stimulated not μm. fixed paraformaldehyde 37 °C, followed methanol permeabilization min −20 °C. Coverslips nonspecific blocked PBS 2% (BSA) 0.2% gelatin. diluted PBS/BSA/gelatin (anti-phospho-Thr453, 1/120; anti-Myc, 1/1000) h, five time PBS. Prior second (biotin-conjugated goat anti-rabbit, 1/1000 fluorescein isothiocyanate-conjugated anti-mouse, 1/100), 4′,6-diamidine dihydrochloride (Roche) concentration 0.2 last enumerate After extensive washes distilled water, mounted Citifluor examined epifluorescence illumination. (20 Tris, 7.5, 10 mmpara-nitrophenyl phosphate, mmMgCl2, dithiothreitol) ∼5 GST/Sp1 equimolar amounts (0.6 μg), (0.3 BSA (0.4 myelin (0.12 GST-ATF2 GST-Elk1 GST-Jun μg) substrates 5 μCi 50 μm[γ-32P]ATP 30 reaction stopped addition Laemmli sample resolved SDS-PAGE. vectors stimulation, immediately buffer. Eighty SDS-PAGE gels transferred onto polyvinylidene difluoride membrane (Immobilon). membranes purified anti-phospho-Thr739 (1/1000) anti-phospho-MAPK (1/5000) anti-total (PEP2 Biotechnology, 1/2000). labeled anti-rabbit horseradish peroxidase-conjugated secondary developed ECL system Inc.). long term transcription. effect found dependent recruitment located proximal (−88/−66) To determine whether direct, EMSA extracts estradiol-inducible (ΔRaf-1:ER) exclusively (21Lenormand ΔRaf-1:ER estradiol. When untreated basal double-stranded encompassing −88/−66-bp detected (Fig. 1A, lane 1). 3) 5), increase observed. complexes, estradiol-stimulated 4) 6), almost entirely disrupted excess unlabeled oligonucleotide, demonstrating complexes contain Sp1-related proteins. when 6) used, part complex B resists competition oligonucleotide. One factor, recruited demonstrate min, anti-Sp1 antibody. Our clearly (present B1 B2) 1B). Supershift anti-Sp3 contained B3 c (data shown). Band cycloheximide, inhibitor synthesis. conditions Because occurred neo-synthesis blocked, postulated could accounted direct investigated Thus, equivalent molar AP-2, (BSA), GST-ATF2, GST-Jun, GST-Elk1, specific substrate SAPK/c-Jun p38/HOG (60Gupta Campbell Derijard Science. 389-393Crossref (1339) 61Livingstone Patel Jones 1785-1797Crossref (476) (62Derijard Hibi Wu Barrett T., Su, Deng Karin 76: 1025-1037Abstract (2957) considered negative controls. (63Hipskind Buscher Baccarini Genes Dev. 8: 1803-1816Crossref (108) 64Hipskind 6219-6231Crossref (137) (65Meloche 63-71Crossref (131) commonly positive strongly GST-E