FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2044147580> ?p ?o ?g. }

• W2044147580 endingPage "29708" @default.
• W2044147580 startingPage "29701" @default.
• W2044147580 abstract "Despite sequence variation, all AP-2 isotypes are capable of activating transcription, which indicates a functional conservation. We used this property to gain a unique insight into the structure and function of the activation motifs of AP-2 family transcription factors. We have precisely localized the activation motif of human AP-2α to amino acids 52–108. Our experiments indicate that similar sequence of amino acids in all AP-2 isotypes exceptDrosophila AP-2α harbor their activation motifs. Within this sequence, fewer than 36 residues are critical for transcription activation. Our comparison studies and site-directed mutagenic analyses show that these critical amino acids are strategically placed within this sequence. These residues are interspersed with nonessential and influential residues that vary in composition and length, indicating a structural flexibility. The Drosophila AP-2α has its partly conserved activation motif in an extended region about twice the length of other AP-2 isotypes. Our results reveal essential elements of the amino acid composition of activators in general and shed new light on the mechanism of transcription activation. Despite sequence variation, all AP-2 isotypes are capable of activating transcription, which indicates a functional conservation. We used this property to gain a unique insight into the structure and function of the activation motifs of AP-2 family transcription factors. We have precisely localized the activation motif of human AP-2α to amino acids 52–108. Our experiments indicate that similar sequence of amino acids in all AP-2 isotypes exceptDrosophila AP-2α harbor their activation motifs. Within this sequence, fewer than 36 residues are critical for transcription activation. Our comparison studies and site-directed mutagenic analyses show that these critical amino acids are strategically placed within this sequence. These residues are interspersed with nonessential and influential residues that vary in composition and length, indicating a structural flexibility. The Drosophila AP-2α has its partly conserved activation motif in an extended region about twice the length of other AP-2 isotypes. Our results reveal essential elements of the amino acid composition of activators in general and shed new light on the mechanism of transcription activation. activation domain DNA-binding domain chloramphenicol acetyltransferase human AP-2 mouse AP-2 Xenopus AP-2 chicken AP-2 Drosophila AP-2 polymerase chain reaction Transcription factors tightly regulate gene expression in response to intra- and extracellular stimuli, and they often play a central role in determining cell fate by controlling the fundamental mechanism of gene transcription. Transcription factor AP-2α regulates the genes involved in a spectrum of important biological functions. Some of the AP-2α-activated genes are p21WAF1/CIP1 (1Zeng Y.X. Somasundaram K. el-Deiry W.S. Nat. Genet. 1997; 15: 78-82Crossref PubMed Scopus (260) Google Scholar), transforming growth factor-α (2Wang D. Shin T.H. Kudlow J.E. J. Biol. Chem. 1997; 272: 14244-14250Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), estrogen receptor (3McPherson L.A. Baichwal V.R. Weigel R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4342-4347Crossref PubMed Scopus (130) Google Scholar), keratinocyte-specific genes (4Leask A. Byrne C. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7948-7952Crossref PubMed Scopus (204) Google Scholar), tyrosine kinase receptor gene c-KIT (5Huang S. Jean D. Luca M. Tainsky M.A. Bar-Eli M. EMBO J. 1998; 17: 4358-4369Crossref PubMed Scopus (228) Google Scholar), HIV type-1 (6Perkins N.D. Agranoff A.B. Duckett C.S. Nabel G.J. J. Virol. 1994; 68: 6820-6823Crossref PubMed Google Scholar), HTLV-I (7Muchardt C. Seeler J.S. Gaynor R.B. New Biol. 1992; 4: 541-550PubMed Google Scholar, 8Muchardt C. Seeler J.S. Nirula A. Gong S. Gaynor R. EMBO J. 1992; 11: 2573-2581Crossref PubMed Scopus (39) Google Scholar), type IV collagenase (9Frisch S.M. Morisaki J.H. Mol. Cell. Biol. 1990; 10: 6524-6532Crossref PubMed Scopus (109) Google Scholar), SV40 enhancer region, human metallothionein gene IIa (10Mitchell P.J. Wang C. Tjian R. Cell. 1987; 50: 847-861Abstract Full Text PDF PubMed Scopus (630) Google Scholar), HER-2/neu (11Bosher J.M. Totty N.F. Hsuan J.J. Williams T. Hurst H.C. Oncogene. 1996; 13: 1701-1707PubMed Google Scholar, 12Bosher J.M. Williams T. Hurst H.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 744-747Crossref PubMed Scopus (208) Google Scholar), insulin-like growth factor-binding protein-5 (13Duan C. Clemmons D.R. J. Biol. Chem. 1995; 270: 24844-24851Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and the dopamine β-hydroxylase gene (14Greco D. Zellmer E. Zhang Z. Lewis E. J. Neurochem. 1995; 65: 510-516Crossref PubMed Scopus (42) Google Scholar). AP-2α also negatively regulates a number of genes, includingMCAM/MUC18 (15Jean D. Gershenwald J.E. Huang S. Luca M. Hudson M.J. Tainsky M.A. Bar-Eli M. J. Biol. Chem. 1998; 273: 16501-16508Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar); c/EBP-α, during adipogenesis; and c-myc (16Gaubatz S. Imhof A. Dosch R. Werner O. Mitchell P. Buettner R. Eilers M. EMBO J. 1995; 14: 1508-1519Crossref PubMed Scopus (178) Google Scholar). Retinoic acid, a developmental morphogen that is involved in vertebrate limb bud pattern formation, induces AP-2α gene expression transiently in the teratocarcinoma cell lines N-Tera-2 (17Luscher B. Mitchell P.J. Williams T. Tjian R. Genes Dev. 1989; 3: 1507-1517Crossref PubMed Scopus (213) Google Scholar,18Williams T. Admon A. Luscher B. Tjian R. Genes Dev. 1988; 2: 1557-1569Crossref PubMed Scopus (450) Google Scholar) and PA-1 (19Buettner R. Kannan P. Imhof A. Bauer R. Yim S.O. Glockshuber R. Van Dyke M.W. Tainsky M.A. Mol. Cell. Biol. 1993; 13: 4174-4185Crossref PubMed Scopus (123) Google Scholar). AP-2α mediates transcriptional activation in response to two signal transduction pathways: the phorbol ester/diacylglycerol-inducible protein kinase C pathway and the cAMP-dependent protein kinase A pathway (20Chiu R. Imagawa M. Imbra R.J. Bockoven J.R. Karin M. Nature. 1987; 329: 648-651Crossref PubMed Scopus (220) Google Scholar, 21Imagawa M. Chiu R. Karin M. Cell. 1987; 51: 251-260Abstract Full Text PDF PubMed Scopus (1030) Google Scholar). Aberration of AP-2 activity profoundly affects the cell and the organism. AP-2α null mice have multiple congenital defects at birth, including defective skin and craniofacial abnormalities (22Zhang J. Hagopian-Donaldson S. Serbedzija G. Elsemore J. Plehn-Dujowich D. McMahon A.P. Flavell R.A. Williams T. Nature. 1996; 381: 238-241Crossref PubMed Scopus (534) Google Scholar, 23Schorle H. Meier P. Buchert M. Jaenisch R. Mitchell P.J. Nature. 1996; 381: 235-238Crossref PubMed Scopus (519) Google Scholar). Mice lacking AP-2β die in early postnatal days from enhanced apoptotic cell death of renal epithelial cells (24Moser M. Pscherer A. Roth C. Becker J. Mucher G. Zerres K. Dixkens C. Weis J. Guay-Woodford L. Buettner R. Fassler R. Genes Dev. 1997; 11: 1938-1948Crossref PubMed Scopus (246) Google Scholar). A growing number of studies attribute abnormal AP-2 activity to cancer progression. Their discoveries include the following: a critical role for AP-2α inras-oncogene-induced transformation of the human teratocarcinoma cells PA-1 (25Kannan P. Buettner R. Chiao P.J. Yim S.O. Sarkiss M. Tainsky M.A. Genes Dev. 1994; 8: 1258-1269Crossref PubMed Scopus (93) Google Scholar); constitutive expression of AP-2α in SV40 large T antigen-immortalized human lung fibroblasts unlike in normal cells, indicating a relationship between AP-2α expression and immortalization (26Huang Y. Domann F.E. Arch. Biochem. Biophys. 1999; 364: 241-246Crossref PubMed Scopus (16) Google Scholar); overexpression of insulin-like growth factor II in rhabdomyosarcoma is caused by AP-2 (27Zhang L. Zhan S. Navid F. Li Q. Choi Y.H. Kim M. Seth P. Helman L.J. Oncogene. 1998; 17: 1261-1270Crossref PubMed Scopus (24) Google Scholar); overexpression of HER-2/neu in breast cancer was induced by AP-2α and AP-2γ (12Bosher J.M. Williams T. Hurst H.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 744-747Crossref PubMed Scopus (208) Google Scholar); regulation of estrogen receptor by AP-2γ in hormone-responsive mammary cancer (3McPherson L.A. Baichwal V.R. Weigel R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4342-4347Crossref PubMed Scopus (130) Google Scholar); AP-2α and AP-2γ regulation of various growth factor signaling pathways that play a role in breast cancer (28Turner B.C. Zhang J. Gumbs A.A. Maher M.G. Kaplan L. Carter D. Glazer P.M. Hurst H.C. Haffty B.G. Williams T. Cancer Res. 1998; 58: 5466-5472PubMed Google Scholar); dysregulation of AP-2α causes aberrant expression of c-KIT and MCAM/MUC18 in malignant melanoma (29Bar-Eli M. J. Cell. Physiol. 1997; 173: 275-278Crossref PubMed Scopus (75) Google Scholar); and AP-2α correlates with low p21 expression, malignant transformation, and tumor progression in cutaneous malignant melanoma (30Karjalainen J.M. Kellokoski J.K. Eskelinen M.J. Alhava E.M. Kosma V.M. J. Clin. Oncol. 1998; 16: 3584-3591Crossref PubMed Scopus (92) Google Scholar). We made a GAL4-hAP-2α fusion protein in which the activation domain (AD)1 of hAP-2α was linked to a heterologous DNA-binding domain (DBD) of GAL4 and found that it retained the oncogenic property of AP-2 (25Kannan P. Buettner R. Chiao P.J. Yim S.O. Sarkiss M. Tainsky M.A. Genes Dev. 1994; 8: 1258-1269Crossref PubMed Scopus (93) Google Scholar). Nontumorigenic PA-1 cells constitutively overexpressing GAL4-hAP-2α induced tumors in nude mice similar to the tumorigenic ras PA-1 cells and hAP-2α-overexpressing PA-1 cells (31Kannan P. Tainsky M.A. Mol. Cell. Biol. 1999; 19: 899-908Crossref PubMed Scopus (53) Google Scholar). These observations suggested that the AD of hAP-2α mediated tumorigenicity. Our studies indicate that the coactivator PC4, which interacts with the AD of hAP-2α suppresses tumorigenicity in ras-transformed cells (31Kannan P. Tainsky M.A. Mol. Cell. Biol. 1999; 19: 899-908Crossref PubMed Scopus (53) Google Scholar). Therefore, a detailed analysis of AD of hAP-2α is crucial to understanding its role in tumorigenicity and to identifying additional factors that interact with this region and participate in transformation. Several forms of AP-2 have been isolated from various species, and they constitute a family of transcription factors. AP-2α has been isolated from human (hAP-2α) (32Mitchell P.J. Timmons P.M. Hebert J.M. Rigby P.W. Tjian R. Genes Dev. 1991; 5: 105-119Crossref PubMed Scopus (498) Google Scholar), murine (mAP-2α) (33Moser M. Pscherer A. Bauer R. Imhof A. Seegers S. Kerscher M. Buettner R. Nucleic Acids Res. 1993; 21: 4844Crossref PubMed Scopus (12) Google Scholar), chicken (cAP-2α) (34Shen H. Wilke T. Ashique A.M. Narvey M. Zerucha T. Savino E. Williams T. Richman J.M. Dev. Biol. 1997; 188: 248-266Crossref PubMed Scopus (114) Google Scholar), Drosophila (dAP-2α) (35Bauer R. McGuffin M.E. Mattox W. Tainsky M.A. Oncogene. 1998; 17: 1911-1922Crossref PubMed Scopus (24) Google Scholar, 36Monge I. Mitchell P.J. Mech Dev. 1998; 76: 191-195Crossref PubMed Scopus (29) Google Scholar) andXenopus (xAP-2α) (37Winning R.S. Shea L.J. Marcus S.J. Sargent T.D. Nucleic Acids Res. 1991; 19: 3709-3714Crossref PubMed Scopus (64) Google Scholar). AP-2β has been isolated from human (hAP-2β) (11Bosher J.M. Totty N.F. Hsuan J.J. Williams T. Hurst H.C. Oncogene. 1996; 13: 1701-1707PubMed Google Scholar), murine (mAP-2β) (38Moser M. Imhof A. Pscherer A. Bauer R. Amselgruber W. Sinowatz F. Hofstadter F. Schule R. Buettner R. Development. 1995; 121: 2779-2788Crossref PubMed Google Scholar), and chicken (cAP-2β) (39Bisgrove D.A. Godbout R. Dev. Dyn. 1999; 214: 195-206Crossref PubMed Scopus (42) Google Scholar). AP-2γ was isolated from human (hAP-2γ) (3McPherson L.A. Baichwal V.R. Weigel R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4342-4347Crossref PubMed Scopus (130) Google Scholar, 11Bosher J.M. Totty N.F. Hsuan J.J. Williams T. Hurst H.C. Oncogene. 1996; 13: 1701-1707PubMed Google Scholar) and murine (mAP-2γ) (40Chazaud C. Oulad-Abdelghani M. Bouillet P. Decimo D. Chambon P. Dolle P. Mech Dev. 1996; 54: 83-94Crossref PubMed Scopus (153) Google Scholar). All forms of AP-2 show striking amino acid sequence conservation at their C termini in the region of the DBD and, as expected, they bind to same-target sequences. A comparison of the DNA-binding sites of hAP-2α and hAP-2γ was made recently, and their consensus-binding site was determined to be (G/C)CCNN(A/C/G)(G/A)G(G/C/T) (41McPherson L.A. Weigel R.J. Nucleic Acids Res. 1999; 27: 4040-4049Crossref PubMed Scopus (96) Google Scholar). The conservation of amino acid sequences occurs relatively less in the N termini, but despite the resulting sequence variation, all forms of AP-2 possess an AD at their N termini. This observation presents a unique opportunity to understand the nature and constitution of the activation motif of AP-2 by comparing the functional role of conserved amino acid sequences. In this report, we have characterized the activation motif of hAP-2α by precisely identifying the amino acids required for its function. The corresponding sequences of other isotypes from various species were also examined to gain insight into the activation property of the AP-2 family of transcription factors. NIH 3T3 and COS-1 cells were grown in modified Eagle's medium with Earl's salts (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hazelton Biologics, Lenexa, KS) and antibiotics at 37 °C in 5% CO2, 95% air. 9117 cells, a subline of PA-1 human teratocarcinoma (42Zeuthen J. Norgaard J.O. Avner P. Fellous M. Wartiovaara J. Vaheri A. Rosen A. Giovanella B.C. Int. J. Cancer. 1980; 25: 19-32Crossref PubMed Scopus (150) Google Scholar), were grown in similar conditions but supplemented with 5% fetal bovine serum. Transient transfections were performed using SuperFect reagent (Qiagen Inc., Valencia, CA) in NIH 3T3 and COS-1 cells and calcium phosphate precipitate (43Graham F.L. Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6498) Google Scholar) in PA-1 cells grown on 100-mm dishes to introduce DNA. The amount of DNA used in all transfections was equalized by the addition of pBluescript DNA. 1 μg of β-galactosidase expression vector pCH110 (Amersham Pharmacia Biotech) was included in each transfection, and the efficiency of transfection was normalized after assaying the β-galactosidase enzyme (44Miller J.H. Schmeissner U. J. Mol. Biol. 1979; 131: 223-248Crossref PubMed Scopus (57) Google Scholar). AP-2 reporter constructs 3× AP-2hMt-CAT and 3× AP-2SV40-CAT contain three 32-base pair synthetic oligonucleotides corresponding to the AP-2 response element harboring sequence of the distal basal-level element of human metallothionein gene IIa or SV40 enhancer region and a HSVtk promoter in the vector pBLCAT2 (45Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 549Google Scholar). Reporter construct G5E1bCAT, a generous gift of Dr. Ptashne (46Sadowski I. Ma J. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Crossref PubMed Scopus (989) Google Scholar) was used to measure the transactivation activity of GAL4-AP-2 fusion constructs. CAT activity in normalized protein was measured by converting [14C]chloramphenicol to mono- and di-acetyl chloramphenicol, essentially as described earlier (47Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar). After partitioning the acetylated forms of chloramphenicol on thin layer chromatography plates, the percentage of conversion was calculated by measuring radioactivity on a Storm analyzer (Molecular Dynamics, Inc., Sunnyvale, CA). The GAL4 DBD and hAP-2α AD containing fusion construct pGAL4-hAP-2α/11–226 was made by inserting amino acids 11–226 of hAP-2α into EcoRI-cut and mung bean nuclease-blunted pSG424 (46Sadowski I. Ma J. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Crossref PubMed Scopus (989) Google Scholar). pGAL4-hAP-2α/11–165 and pGAL4-hAP-2α/11–121 were derived from this construct by deleting the sequence after SmaI and BamHI, respectively. To make further sequential deletions, plasmid pGAL4-hAP-2α/11–226 was linearized at the BamHI site, which cuts after amino acid 121, and Bal31 nuclease was used to remove nucleotides for 1–5 min. After exonuclease digestion, the nonessential C-terminal amino acids 122–226 were removed using SacI, which cuts before the three stop codons. One stop codon exists in each reading frame to ensure termination of protein translation in all reading frames. The plasmids were subjected to dideoxy sequencing to verify sequential deletions. To make precise deletions in the activation motif polymerase chain reaction (PCR) amplification method was used with a forward oligo containing a BamHI site (5′-GCTACGGATCCXn-3′) and a reverse oligo containing a KpnI site (5′-ACGTTGGTACCXn-3′).Xn refers to 15–20 nucleotides from AP-2 isotypes at the desired end points. After PCR amplification, the fragments were digested with BamHI and KpnI and cloned adjacent to the GAL4 DBD in the vector pSG424. Forward oligo containing aSalI site (5′-ACGTGCACXn-3′) was employed to amplify the hAP-2γ and dAP-2α to avoid complications of an internal BamHI site in the desired regions in these isotypes. The reading frame was maintained for each clone to ensure the fusion of GAL4 DBD and AP-2. The mutant clones GAL4-cAP-2β 53–114 N85D and GAL4-cAP-2β 53–114 Q89R were inadvertently obtained during the preparation of GAL4-cAP-2β 53–114, because of the inherent property of PCR amplification. Other site-directed alterations were made in full-length AP-2 molecules using two complementary oligos with the desired change and the QuickChange site-directed mutagenesis kit (Stratagene Cloning Systems, La Jolla, CA). All constructs were subjected to double-stranded DNA sequencing to verify the clone and/or to ensure the proper reading frame. 5 μg of GAL4-AP-2 fusion protein expression plasmid DNA was transfected into COS-1 cells using SuperFect reagent (Qiagen). After 48 h the cells were harvested and suspended in lysis buffer (1.5% Triton X-100, 2 mmphenylmethylsulfonyl fluoride, 0.5 mm EDTA, pH 8.0, aprotinin 90 μg/ml, leupeptin 50 μg/ml, and benzamidine HCl 20 μg/ml in phosphate-buffered saline, pH 7.2). The cells were lysed by three cycles of freezing and thawing, rocked for 30 min at 4 °C, and spun for 30 min at 14,000 rpm. The supernatant containing the crude protein extract (50 μg) was separated on a 14% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (NEN Life Science Products). The membrane was probed with anti-GAL4-DBD polyclonal antibody made from rabbit (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The signals were detected using anti-rabbit IgG conjugated with horseradish peroxidase and ECL kit (Amersham Pharmacia Biotech). Isotypes of AP-2 transcription factor AP-2α, AP-2β, and/or AP-2γ have been identified from human, murine, chicken, Drosophila, and Xenopus systems (3McPherson L.A. Baichwal V.R. Weigel R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4342-4347Crossref PubMed Scopus (130) Google Scholar, 11Bosher J.M. Totty N.F. Hsuan J.J. Williams T. Hurst H.C. Oncogene. 1996; 13: 1701-1707PubMed Google Scholar, 32Mitchell P.J. Timmons P.M. Hebert J.M. Rigby P.W. Tjian R. Genes Dev. 1991; 5: 105-119Crossref PubMed Scopus (498) Google Scholar, 33Moser M. Pscherer A. Bauer R. Imhof A. Seegers S. Kerscher M. Buettner R. Nucleic Acids Res. 1993; 21: 4844Crossref PubMed Scopus (12) Google Scholar, 34Shen H. Wilke T. Ashique A.M. Narvey M. Zerucha T. Savino E. Williams T. Richman J.M. Dev. Biol. 1997; 188: 248-266Crossref PubMed Scopus (114) Google Scholar, 35Bauer R. McGuffin M.E. Mattox W. Tainsky M.A. Oncogene. 1998; 17: 1911-1922Crossref PubMed Scopus (24) Google Scholar, 36Monge I. Mitchell P.J. Mech Dev. 1998; 76: 191-195Crossref PubMed Scopus (29) Google Scholar, 38Moser M. Imhof A. Pscherer A. Bauer R. Amselgruber W. Sinowatz F. Hofstadter F. Schule R. Buettner R. Development. 1995; 121: 2779-2788Crossref PubMed Google Scholar, 39Bisgrove D.A. Godbout R. Dev. Dyn. 1999; 214: 195-206Crossref PubMed Scopus (42) Google Scholar, 40Chazaud C. Oulad-Abdelghani M. Bouillet P. Decimo D. Chambon P. Dolle P. Mech Dev. 1996; 54: 83-94Crossref PubMed Scopus (153) Google Scholar). All forms of AP-2 show strong amino acid sequence conservation near their C termini beginning with the glutamine amino acid. The position of the glutamine residue in hAP-2α is 209. As shown in Fig. 1, the amino acid sequence of hAP-2β is 93% and that of hAP-2γ is 84%, both of which are similar to hAP-2α. Their DBDs are situated in this region, and, as expected, they bind to same target sequences. The sequence is relatively less conserved at their N termini where they contain their ADs. The hAP-2β and hAP-2γ isotypes are 66 and 63% similar to hAP-2α, respectively, in this region. Interestingly, isotype-specific sequence conservation is retained across many species, especially in human, mouse, and chicken. Mouse and chicken AP-2α and AP-2β isotypes are very similar to their human counterparts, mAP-2γ is very similar to hAP-2γ, and xAP-2 is very similar to hAP-2α. dAP-2α, however, is significantly less conserved. All are better conserved in the region of their DBDs than their N termini. Irrespective of sequence variation at their N termini, all are able to activate transcription. We used this property to examine the sequences responsible for transcription activation. We cloned AP-2 isotypes that showed variation in sequence at their N termini in a mammalian expression vector pSG5 under the control of an SV40 promoter to have an identical backbone. An AP-2 reporter construct 3× AP-2hMt-CAT, containing three AP-2-binding sites derived from the human metallothionein gene IIa promoter, was used to measure AP-2 activity in NIH 3T3 cells. Expression plasmids of AP-2 were cotransfected with the reporter construct, and AP-2 activity was determined. As shown in Fig. 2, hAP-2α activated transcription 2-fold above the endogenous level when 1 μg of its expression plasmid DNA was transfected. The dAP-2α was the strongest activator with about 10-fold induction. In these experiments, xAP-2α activated transcription about 7-fold, cAP-2α 2-fold, mAP-2β 3-fold, cAP-2β 4-fold, hAP-2γ 4-fold, and mAP-2γ 6-fold. We examined their activities using a different AP-2 reporter construct, 3× AP-2SV40-CAT, with AP-2-binding sites derived from an SV40 promoter and found that the pattern of their activation was similar (data not shown). We tested their activity in the human teratocarcinoma cell line PA-1 using 3× AP-2SV40-CAT and observed similar activation of transcription by AP-2 isotypes (Fig. 2). mAP-2β and cAP-2β activation was nearly 2-fold stronger than that observed with 3× AP-2hMT-CAT in NIH 3T3 cells. These results indicate that all AP-2 are capable of activating transcription, albeit with different levels of efficiency. Our next strategy was to narrow the ADs of AP-2 isotypes to the minimal sequences necessary and study their amino acid conservation. First, we selected the hAP-2α, because some preliminary characterization has been done on the AD of this protein (25Kannan P. Buettner R. Chiao P.J. Yim S.O. Sarkiss M. Tainsky M.A. Genes Dev. 1994; 8: 1258-1269Crossref PubMed Scopus (93) Google Scholar, 48Williams T. Tjian R. Genes Dev. 1991; 5: 670-682Crossref PubMed Scopus (443) Google Scholar) and performed a fine structure analysis to precisely identify the minimal amino acid sequence necessary to activate transcription. Later, we used this information to identify sequences in other AP-2 isotypes to verify their activation and to study how the activation function was conserved. The N terminus of hAP-2α that harbors its activation domain was sequentially deleted and fused to the heterologous GAL4 DBD. As shown in Fig.3 A, the N-terminal 11–226 amino acids are able to activate transcription from the GAL4 reporter construct G5E1bCAT. This region of AP-2 contains the entire AD and the N-terminal region of DBD as previously characterized (48Williams T. Tjian R. Genes Dev. 1991; 5: 670-682Crossref PubMed Scopus (443) Google Scholar). The transactivation activity stimulated by this region is arbitrarily set to 100%. Transcription activation by amino acids 11–165 was reduced by about one fourth. Interestingly, the removal of 44 additional amino acids from the C terminus, as in 11–121, restored full transcription. This observation suggests that the region between amino acids 122 and 165 has a negative effect on hAP-2α activation. Amino acids between 11 and 118 and between 11 and 117 activated transcription similar to amino acids 11–121, indicating that the central activation motif is between amino acids 11 and 117. A slight reduction in transcription was seen with the construct 11–107. Removal of two additional amino acids, as in 11–105, reduced transcription dramatically, indicating that glycine at position 106, leucine at position 107, or both are essential for AP-2 transcription activation. The constructs containing amino acids between 11 and 105, 11 and 103, 11 and 102, and 11 and 93 retained nearly 10% of the ability to activate transcription when compared with GAL4-hAP-2α/11–226. Further removal of amino acids, as in 11–76 or 11–30, near completely abolished transcription activation. The transcriptional activation of these GAL4-hAP-2α fusion constructs was similar in COS-1 cells (not shown). The expression of these fusion constructs is comparable in these cells, and some of them are shown in Fig. 3 B. An earlier study (48Williams T. Tjian R. Genes Dev. 1991; 5: 670-682Crossref PubMed Scopus (443) Google Scholar) and our preliminary observation indicate that removal of the N-terminal 50 amino acids does not affect the activation function of hAP-2α. To precisely find the N-terminal boundary of the sequence that activates transcription, we prepared DNA fragments that varied by one to a few amino acids. These fragments were linked to the GAL4 DBD, and their activation properties were determined. The amino acids between 51 and 117 and between 52 and 117 showed near full efficiency of activation indicating that the central core of the activation motif resides in this region. The activity of the construct containing the amino acids between 53 and 117 was reduced more than 6-fold, indicating that the aspartic acid at position 52 is the first critical residue at the N-terminus of hAP-2α. Likewise, the construct containing amino acids between 55 and 117 also did not significantly activate transcription. We progressively narrowed the distal portion of the hAP-2α AD to more precisely identify the last critical residue of activation. As shown above in the sequential deletion experiments, the constructs containing the amino acids 11–107 and 11–117 had 82 and 109% activity, respectively, but amino acids 11–105 were unable to activate transcription. Constructs containing the amino acids 52–117, 52–114, 52–110, and 52–108 activated transcription significantly. The 107% transcription activity stimulated by the construct containing amino acids between 52 and 108 indicates that the central minimal motif of hAP-2α resides within this sequence. A construct containing the amino acids 51–106 lost activation properties dramatically, indicating that leucine at 107 is critical for activation. The residues at positions 107 and 108 are both leucines. The amino acids 51–77 fused to GAL4-DBD do not activate transcription. However, constructs containing the amino acids 6–77 and 20–77 activated about one-eighth activity. The weak activation by the amino acids 6–77 and 20–77 when compared with the relatively no activation by the amino acids 51–77 suggest that the region between amino acids 20 and 50 have a weak intrinsic activation property. This region also positively elevates the activity of hAP-2α when the constructs 20–110 and 51–110 are compared. As summarized in Fig.4 A, the amino acid sequence between the critical aspartic acid at position 52 and the leucines at positions 107 and 108 harbors the central activation motif of hAP-2α, which can significantly activate transcription. There are two regions, one between amino acids 20 and 51 and another between 165 and 226, that positively affect hAP-2α activity. The region between amino acids 122 and 165 has a negative effect. To gain a detailed understanding of the structure and function of the activation motifs of all AP-2 family transcription factors, we identified the region corresponding to the amino acids between 52 and 108 of hAP-2α in other AP-2 isotypes of various species. We PCR amplified the identified regions, linked them to GAL4 DBD, and tested their ability to activate transcription from a GAL4 target sequence. In such experiments, the amino acid sequences will demonstrate their capability of transcriptional activation, and their sequence variation will serve as a natural source of mutations that can be used for analysis. If two forms of AP-2 have 100% identity in this region, such as hAP-2α and mAP-2α or hAP-2β and mAP-2β, only one of them was selected for examination. A comparison of the amino acid sequences of selected AP-2 isotypes and the efficiency of transcription activation by select regions are shown in Fig.4 B. The selected sequences of AP-2 isotypes except dAP-2α are capable of activating transcription albeit with varied efficiency in both NIH 3T3 and PA-1 cells. Their expression is comparable in COS-1 cells (Fig. 4 C). The efficiency of their transcription activation, although not identical, is comparable with the relative activities of corresponding full-length AP-2 isotypes from AP-2 target sequences (Fig. 1). These results demonstrate" @default.
• W2044147580 created "2016-06-24" @default.
• W2044147580 creator A5008379792 @default.
• W2044147580 creator A5036204735 @default.
• W2044147580 creator A5036753038 @default.
• W2044147580 creator A5050473909 @default.
• W2044147580 creator A5065937259 @default.
• W2044147580 date "2000-09-01" @default.
• W2044147580 modified "2023-10-09" @default.
• W2044147580 title "Characterization of the Activation Domains of AP-2 Family Transcription Factors" @default.
• W2044147580 cites W135917912 @default.
• W2044147580 cites W1495868618 @default.
• W2044147580 cites W1658225810 @default.
• W2044147580 cites W1964811259 @default.
• W2044147580 cites W1965372133 @default.
• W2044147580 cites W1969591502 @default.
• W2044147580 cites W1970124231 @default.
• W2044147580 cites W1975118160 @default.
• W2044147580 cites W1977520190 @default.
• W2044147580 cites W1979647722 @default.
• W2044147580 cites W1982288359 @default.
• W2044147580 cites W1986636337 @default.
• W2044147580 cites W1989429141 @default.
• W2044147580 cites W1993214904 @default.
• W2044147580 cites W1997973068 @default.
• W2044147580 cites W2006045768 @default.
• W2044147580 cites W2008841836 @default.
• W2044147580 cites W2017243155 @default.
• W2044147580 cites W2017867661 @default.
• W2044147580 cites W2021052173 @default.
• W2044147580 cites W2024919800 @default.
• W2044147580 cites W2026237709 @default.
• W2044147580 cites W2029506276 @default.
• W2044147580 cites W2029787649 @default.
• W2044147580 cites W2034612795 @default.
• W2044147580 cites W2045467759 @default.
• W2044147580 cites W2045545244 @default.
• W2044147580 cites W2045622916 @default.
• W2044147580 cites W2046570924 @default.
• W2044147580 cites W2047207093 @default.
• W2044147580 cites W2053838398 @default.
• W2044147580 cites W2061689243 @default.
• W2044147580 cites W2065579085 @default.
• W2044147580 cites W2068599501 @default.
• W2044147580 cites W2079027524 @default.
• W2044147580 cites W2079584632 @default.
• W2044147580 cites W2083755346 @default.
• W2044147580 cites W2083869788 @default.
• W2044147580 cites W2092356591 @default.
• W2044147580 cites W2101816208 @default.
• W2044147580 cites W2103238195 @default.
• W2044147580 cites W2106882534 @default.
• W2044147580 cites W2108754630 @default.
• W2044147580 cites W2112365969 @default.
• W2044147580 cites W2154024738 @default.
• W2044147580 cites W2163408738 @default.
• W2044147580 cites W2239490943 @default.
• W2044147580 cites W2317539371 @default.
• W2044147580 cites W2330626449 @default.
• W2044147580 cites W38542220 @default.
• W2044147580 doi "https://doi.org/10.1074/jbc.m000931200" @default.
• W2044147580 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10899156" @default.
• W2044147580 hasPublicationYear "2000" @default.
• W2044147580 type Work @default.
• W2044147580 sameAs 2044147580 @default.
• W2044147580 citedByCount "58" @default.
• W2044147580 countsByYear W20441475802012 @default.
• W2044147580 countsByYear W20441475802013 @default.
• W2044147580 countsByYear W20441475802014 @default.
• W2044147580 countsByYear W20441475802015 @default.
• W2044147580 countsByYear W20441475802016 @default.
• W2044147580 countsByYear W20441475802019 @default.
• W2044147580 countsByYear W20441475802021 @default.
• W2044147580 countsByYear W20441475802022 @default.
• W2044147580 countsByYear W20441475802023 @default.
• W2044147580 crossrefType "journal-article" @default.
• W2044147580 hasAuthorship W2044147580A5008379792 @default.
• W2044147580 hasAuthorship W2044147580A5036204735 @default.
• W2044147580 hasAuthorship W2044147580A5036753038 @default.
• W2044147580 hasAuthorship W2044147580A5050473909 @default.
• W2044147580 hasAuthorship W2044147580A5065937259 @default.
• W2044147580 hasBestOaLocation W20441475801 @default.
• W2044147580 hasConcept C104317684 @default.
• W2044147580 hasConcept C138885662 @default.
• W2044147580 hasConcept C179926584 @default.
• W2044147580 hasConcept C185592680 @default.
• W2044147580 hasConcept C41895202 @default.
• W2044147580 hasConcept C54355233 @default.
• W2044147580 hasConcept C70721500 @default.
• W2044147580 hasConcept C86339819 @default.
• W2044147580 hasConcept C86803240 @default.
• W2044147580 hasConcept C95444343 @default.
• W2044147580 hasConceptScore W2044147580C104317684 @default.
• W2044147580 hasConceptScore W2044147580C138885662 @default.
• W2044147580 hasConceptScore W2044147580C179926584 @default.
• W2044147580 hasConceptScore W2044147580C185592680 @default.
• W2044147580 hasConceptScore W2044147580C41895202 @default.
• W2044147580 hasConceptScore W2044147580C54355233 @default.

• next