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An Extended α-Helix and Specific Amino Acid Residues Opposite the DNA-binding Surface of the cAMP Response Element Binding Protein Basic Domain Are Important for Human T Cell Lymphotropic Retrovirus Type I Tax Binding

1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês

10.1074/jbc.273.42.27339

ISSN

1083-351X

Autores

Yong Tang, Feng Tie, Imre Boros, Robert Harrod, Mark Glover, Chou‐Zen Giam,

Tópico(s)

Vector-Borne Animal Diseases

Resumo

The human T cell lymphotropic retrovirus type I (HTLV-I) trans-activator, Tax, interacts specifically with the basic-domain/leucine-zipper (bZip) protein, cAMP response element binding protein (CREB), bound to the viral Tax-responsive element consisting of three imperfect 21-base pair repeats, each with a cAMP response element core flanked by G/C-rich sequences. Here, the minimal CREB-bZip necessary for Tax binding is shown to be composed of amino acid residues 280–341. The Tax-CREB interaction involves an uninterrupted and extended α-helix in CREB that spans most of its basic domain to include amino acid residues localized to the NH2 terminus of the DNA binding region. Mutational analyses indicate that three residues, Arg284, Met291, and Glu299 unique to this region of the CREB/activating transcription factor-1 subfamily of bZip proteins, constitute the contact surface for Tax. Amino acid substitutions in these positions had little impact on CREB-bZip binding to DNA but abrogated its binding to Tax. Each of the contact residues for Tax are spaced approximately two helical turns apart on the side of the bZip helix directly opposite to that of the invariant DNA-binding residues. Molecular modeling reveals the Tax-contact residues to be near the minor groove of the G/C-rich DNA in the 21-base pair repeat. They most likely position Tax for minor groove contact with the G/C-rich sequences. The human T cell lymphotropic retrovirus type I (HTLV-I) trans-activator, Tax, interacts specifically with the basic-domain/leucine-zipper (bZip) protein, cAMP response element binding protein (CREB), bound to the viral Tax-responsive element consisting of three imperfect 21-base pair repeats, each with a cAMP response element core flanked by G/C-rich sequences. Here, the minimal CREB-bZip necessary for Tax binding is shown to be composed of amino acid residues 280–341. The Tax-CREB interaction involves an uninterrupted and extended α-helix in CREB that spans most of its basic domain to include amino acid residues localized to the NH2 terminus of the DNA binding region. Mutational analyses indicate that three residues, Arg284, Met291, and Glu299 unique to this region of the CREB/activating transcription factor-1 subfamily of bZip proteins, constitute the contact surface for Tax. Amino acid substitutions in these positions had little impact on CREB-bZip binding to DNA but abrogated its binding to Tax. Each of the contact residues for Tax are spaced approximately two helical turns apart on the side of the bZip helix directly opposite to that of the invariant DNA-binding residues. Molecular modeling reveals the Tax-contact residues to be near the minor groove of the G/C-rich DNA in the 21-base pair repeat. They most likely position Tax for minor groove contact with the G/C-rich sequences. human T cell lymphotropic retrovirus type I activating transcription factor basic domain-leucine zipper CREB binding protein CAAT/enhancer binding protein cAMP response element cAMP response element binding protein glutathioneS-transferase trans-activator from the pX region electrophoretic mobility shift assays polymerase chain reaction amino acid base pair(s). The human T lymphotropic retrovirus type I (HTLV-I)1 trans-activator, Tax, stimulates viral transcription via three imperfect 21-bp repeat DNA elements in the HTLV-I U3 region (1Cann A.J. Irvin S.Y. Chen Fields B.N. Virology. Lippincott-Raven, Philadelphia1996: 1849-1880Google Scholar, 2Paskalis H. Felber B.K. Pavlakis G.N. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6558-6562Crossref PubMed Scopus (105) Google Scholar, 3Rosen C.A. Park R. Sodroski J.G. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4919-4923Crossref PubMed Scopus (46) Google Scholar, 4Brady J. Jeang K.T. Duvall J. Khoury G. J. Virol. 1987; 61: 2175-2181Crossref PubMed Google Scholar, 5Fujisawa J. Toita M. Yoshida M. J. Virol. 1989; 63: 3234-3239Crossref PubMed Google Scholar, 6Giam C.Z. Xu Y.L. J. Biol. Chem. 1989; 264: 15236-15241Abstract Full Text PDF PubMed Google Scholar). Each of the viral 21-bp-repeats contains a cAMP responseelement (CRE) core flanked by 5′ G-rich and 3′ C-rich sequences. The 21-bp repeats, in collaboration with the cellular transcription factors, cAMP responseelement binding protein (CREB), CREB/activating transcriptionfactor 1 (ATF-1) heterodimer, and to a lesser extent, ATF-1 homodimer, form nucleoprotein complexes uniquely capable of recruiting Tax into ternary complexes that mediate trans-activation (7Zhao L.J. Giam C.Z. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11445-11449Crossref PubMed Scopus (168) Google Scholar, 8Zhao L.J. Giam C.Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7070-7074Crossref PubMed Scopus (296) Google Scholar, 9Suzuki T. Fujisawa J.I. Toita M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 610-614Crossref PubMed Scopus (244) Google Scholar, 10Goren I. Semmes O.J. Jeang K.T. Moelling K. J. Virol. 1995; 69: 5806-5811Crossref PubMed Google Scholar, 11Bantignies F. Rousset R. Desbois C. Jalinot P. Mol. Cell. Biol. 1996; 16: 2174-2182Crossref PubMed Scopus (53) Google Scholar, 12Shnyreva M. Munder T. J. Virol. 1996; 70: 7478-7484Crossref PubMed Google Scholar). Whereas CREB binds to the CRE irrespective of DNA sequence context, the stable assembly of Tax, CREB, and DNA into a ternary complex and Tax-mediated trans-activation require the CRE and the 5′ G-rich and 3′ C-rich flanking sequences present in the viral 21-bp repeats (11Bantignies F. Rousset R. Desbois C. Jalinot P. Mol. Cell. Biol. 1996; 16: 2174-2182Crossref PubMed Scopus (53) Google Scholar, 12Shnyreva M. Munder T. J. Virol. 1996; 70: 7478-7484Crossref PubMed Google Scholar, 13Derse D. J. Virol. 1987; 61: 2462-2471Crossref PubMed Google Scholar, 14Montagne J. Beraud C. Crenon I. Lombard Platet G. Gazzolo L. Sergeant A. Jalinot P. EMBO J. 1990; 9: 957-964Crossref PubMed Scopus (55) Google Scholar, 15Paca Uccaralertkun S. Zhao L.J. Adya N. Cross J.V. Cullen B.R. Boros I.M. Giam C.Z. Mol. Cell. Biol. 1994; 14: 456-462Crossref PubMed Google Scholar, 16Yin M.J. Gaynor R.B. Mol. Cell. Biol. 1996; 16: 3156-3168Crossref PubMed Scopus (83) Google Scholar, 17Yin M.J. Paulssen E. Seeler J. Gaynor R.B. J. Virol. 1995; 69: 6209-6218Crossref PubMed Google Scholar, 18Boros I.M. Tie F. Giam C.Z. Virology. 1995; 214: 207-214Crossref PubMed Scopus (17) Google Scholar).In vitro selection also indicates that DNA sequences bound preferentially by the Tax/CREB complex contain the CRE motif flanked by exceedingly long stretches of 5′ G-rich or 3′ C-rich sequences and bear striking resemblance to the HTLV-I 21-bp repeats (15Paca Uccaralertkun S. Zhao L.J. Adya N. Cross J.V. Cullen B.R. Boros I.M. Giam C.Z. Mol. Cell. Biol. 1994; 14: 456-462Crossref PubMed Google Scholar). These results demonstrate directly that HTLV-I Tax is evolved principally toward activating transcription from the viral cis regulatory element, the HTLV-I 21-bp repeats.The molecular basis for the DNA sequence specificity of Tax trans-activation is not well understood. DNase I footprinting and methylation interference performed on the Tax·CREB·21-bp-repeat complex revealed no protein protection of the G-rich and C-rich sequences (15Paca Uccaralertkun S. Zhao L.J. Adya N. Cross J.V. Cullen B.R. Boros I.M. Giam C.Z. Mol. Cell. Biol. 1994; 14: 456-462Crossref PubMed Google Scholar). Further, naturally occurring or genetically engineered single nucleotide substitutions in the G-rich and C-rich sequences do not affect their function (6Giam C.Z. Xu Y.L. J. Biol. Chem. 1989; 264: 15236-15241Abstract Full Text PDF PubMed Google Scholar). Interestingly, recent data indicate that Tax may be involved in contacting the minor groove of the G/C-rich sequences (19Lenzmeier B.A. Giebler H.A. Nyborg J.K. Mol. Cell. Biol. 1998; 18: 721-731Crossref PubMed Google Scholar).The specificity for the flanking sequences requires a specific interaction between Tax and the basic region of CREB (20Adya N. Zhao L.J. Huang W. Boros I. Giam C.Z. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5642-5646Crossref PubMed Scopus (83) Google Scholar). Tax interacts with CREB via the latter's basic domain and amino acid residues in its immediate vicinity (11Bantignies F. Rousset R. Desbois C. Jalinot P. Mol. Cell. Biol. 1996; 16: 2174-2182Crossref PubMed Scopus (53) Google Scholar, 12Shnyreva M. Munder T. J. Virol. 1996; 70: 7478-7484Crossref PubMed Google Scholar, 20Adya N. Zhao L.J. Huang W. Boros I. Giam C.Z. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5642-5646Crossref PubMed Scopus (83) Google Scholar, 21Yin M.J. Gaynor R.B. J. Mol. Biol. 1996; 264: 20-31Crossref PubMed Scopus (47) Google Scholar, 22Perini G. Wagner S. Green M.R. Nature. 1995; 376: 602-605Crossref PubMed Scopus (135) Google Scholar). Like CREB, Tax functions as a dimer (22Perini G. Wagner S. Green M.R. Nature. 1995; 376: 602-605Crossref PubMed Scopus (135) Google Scholar, 23Tie F. Adya N. Greene W.C. Giam C.Z. J. Virol. 1996; 70: 8368-8374Crossref PubMed Google Scholar). Analyses of Tax mutants indicate that the CREB-binding domain is located in the NH2 terminus, while the subunit dimerization domain resides in the middle section of Tax (10Goren I. Semmes O.J. Jeang K.T. Moelling K. J. Virol. 1995; 69: 5806-5811Crossref PubMed Google Scholar, 23Tie F. Adya N. Greene W.C. Giam C.Z. J. Virol. 1996; 70: 8368-8374Crossref PubMed Google Scholar, 24Adya N. Giam C.Z. J. Virol. 1995; 69: 1834-1841Crossref PubMed Google Scholar).CREB and ATFs are prototypic bZip proteins noted for the common basic domain-leucine zipper structure responsible for sequence specific DNA recognition and intersubunit protein-protein interaction, respectively (25Hai T.W. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (753) Google Scholar). Many members of the bZip family, including the mammalian CREB, ATF, c-Jun/c-Fos, and yeast GCN4, bind either the 7-bp AP-1 motif (TGA(C/G)TCA) or the related CRE motif (TGACGTCA). Remarkably, a third group of the families whose members include NF-IL6 (also known as C/EBP-β) and C/EBP, bind a set of DNA elements (T(T/G)NNGNAA(T/G) and CCAAT box, respectively) whose sequences differ significantly from the AP-1 site and CRE.CREB becomes phosphorylated and activated as a function of the cAMP- or Ca2+-mediated signaling process (26Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2041) Google Scholar, 27Gonzalez G.A. Yamamoto K.K. Fischer W.H. Karr D. Menzel P. Biggs W. Vale W.W. Montminy M.R. Nature. 1989; 337: 749-752Crossref PubMed Scopus (644) Google Scholar, 28Sun P. Lou L. Maurer R.A. J. Biol. Chem. 1996; 271: 3066-3073Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Both protein kinase A and Ca2+/calmodulin-dependent kinases (I and IV) have been shown to phosphorylate CREB at Ser133to activate CRE/CREB-mediated transcription (26Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2041) Google Scholar, 27Gonzalez G.A. Yamamoto K.K. Fischer W.H. Karr D. Menzel P. Biggs W. Vale W.W. Montminy M.R. Nature. 1989; 337: 749-752Crossref PubMed Scopus (644) Google Scholar, 28Sun P. Lou L. Maurer R.A. J. Biol. Chem. 1996; 271: 3066-3073Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). A 265-kDa protein called CBP (CREB binding protein), and its cellular homologue, p300, specifically bind the Ser133-phosphorylated form of CREB (29Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1758) Google Scholar) and function as transcriptional co-activators of CREB (30Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1279) Google Scholar). Kwoket al. (31Kwok R.P. Laurance M.E. Lundblad J.R. Goldman P.S. Shih H. Connor L.M. Marriott S.J. Goodman R.H. Nature. 1996; 380: 642-646Crossref PubMed Scopus (308) Google Scholar) have shown recently that Tax interacts directly with the CBP and p300. These data have recently been confirmed and extended (32Giebler H.A. Loring J.E. van Orden K. Colgin M.A. Garrus J.E. Escudero K.W. Brauweiler A. Nyborg J.K. (2) Mol. Cell. Biol. 1997; 17: 5156-5164Crossref PubMed Scopus (164) Google Scholar). Indeed, in in vitro protein/DNA binding reactions, the inclusion of CBP/p300 results in a highly stable quaternary nucleoprotein complex containing the 21-bp repeat/CREB/Tax/CBP-p300 (see “Results” and Ref. 32Giebler H.A. Loring J.E. van Orden K. Colgin M.A. Garrus J.E. Escudero K.W. Brauweiler A. Nyborg J.K. (2) Mol. Cell. Biol. 1997; 17: 5156-5164Crossref PubMed Scopus (164) Google Scholar). In essence, Tax functions as a virus-specific link to connect the transcriptional co-activator, CBP/p300, and possibly other cellular transcription factors in a signal-independent manner to CREB/ATF-1 assembled on the viral 21-bp repeats. This allows HTLV-I viral gene expression to proceed in the absence of cellular activation.X-ray structures of the bZip domain of GCN4 and the bZip heterodimer of c-Jun/c-Fos indicate that the dimerization of bZip proteins results from a coiled-coil structure composed of two intertwined α-helices, each derived from one subunit of the homo- or heterodimer (33Ellenberger T.E. Brandl C.J. Struhl K. Harrison S.C. Cell. 1992; 71: 1223-1237Abstract Full Text PDF PubMed Scopus (813) Google Scholar, 34Konig P. Richmond T.J. J. Mol. Biol. 1993; 233: 139-154Crossref PubMed Scopus (259) Google Scholar, 35Glover J.N. Harrison S.C. Nature. 1995; 373: 257-261Crossref PubMed Scopus (663) Google Scholar, 36Keller W. Konig P. Richmond T.J. J. Mol. Biol. 1995; 254: 657-667Crossref PubMed Scopus (162) Google Scholar). The leucine zipper and the basic domain form a continuous, uninterrupted α-helix. The basic domains extend from the leucine zipper like two prongs of a fork to engage DNA in the major groove (33Ellenberger T.E. Brandl C.J. Struhl K. Harrison S.C. Cell. 1992; 71: 1223-1237Abstract Full Text PDF PubMed Scopus (813) Google Scholar, 34Konig P. Richmond T.J. J. Mol. Biol. 1993; 233: 139-154Crossref PubMed Scopus (259) Google Scholar, 35Glover J.N. Harrison S.C. Nature. 1995; 373: 257-261Crossref PubMed Scopus (663) Google Scholar, 36Keller W. Konig P. Richmond T.J. J. Mol. Biol. 1995; 254: 657-667Crossref PubMed Scopus (162) Google Scholar). Many of the invariant amino acid residues (boldface letters in Fig. 1) in the basic domain are responsible for sequence-specific contacts with DNA (33Ellenberger T.E. Brandl C.J. Struhl K. Harrison S.C. Cell. 1992; 71: 1223-1237Abstract Full Text PDF PubMed Scopus (813) Google Scholar, 34Konig P. Richmond T.J. J. Mol. Biol. 1993; 233: 139-154Crossref PubMed Scopus (259) Google Scholar, 35Glover J.N. Harrison S.C. Nature. 1995; 373: 257-261Crossref PubMed Scopus (663) Google Scholar, 36Keller W. Konig P. Richmond T.J. J. Mol. Biol. 1995; 254: 657-667Crossref PubMed Scopus (162) Google Scholar). In this study, we show that an α-helix extending beyond the DNA-binding domain of CREB bZip is required for Tax binding. Along this extended helix, three amino acid (aa) residues, Arg284, Met291, and Glu299, located on the opposing side of the invariant DNA recognition residues form the contact surface for Tax. The Tax-contact residues span a distance of approximately 23 Å along most of the length of the basic domain and beyond. Interestingly, these three amino acid residues point away from the major groove of the DNA and are posed adjacent to the minor groove of the G/C-rich sequences flanking the CRE motif, which we and others have shown to be critical for Tax binding (11Bantignies F. Rousset R. Desbois C. Jalinot P. Mol. Cell. Biol. 1996; 16: 2174-2182Crossref PubMed Scopus (53) Google Scholar, 12Shnyreva M. Munder T. J. Virol. 1996; 70: 7478-7484Crossref PubMed Google Scholar, 15Paca Uccaralertkun S. Zhao L.J. Adya N. Cross J.V. Cullen B.R. Boros I.M. Giam C.Z. Mol. Cell. Biol. 1994; 14: 456-462Crossref PubMed Google Scholar, 19Lenzmeier B.A. Giebler H.A. Nyborg J.K. Mol. Cell. Biol. 1998; 18: 721-731Crossref PubMed Google Scholar). Our data lend further support to the notion that Tax is involved in minor groove DNA contact with the G/C-rich flanking sequences after its binding to CREB. The human T lymphotropic retrovirus type I (HTLV-I)1 trans-activator, Tax, stimulates viral transcription via three imperfect 21-bp repeat DNA elements in the HTLV-I U3 region (1Cann A.J. Irvin S.Y. Chen Fields B.N. Virology. Lippincott-Raven, Philadelphia1996: 1849-1880Google Scholar, 2Paskalis H. Felber B.K. Pavlakis G.N. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6558-6562Crossref PubMed Scopus (105) Google Scholar, 3Rosen C.A. Park R. Sodroski J.G. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4919-4923Crossref PubMed Scopus (46) Google Scholar, 4Brady J. Jeang K.T. Duvall J. Khoury G. J. Virol. 1987; 61: 2175-2181Crossref PubMed Google Scholar, 5Fujisawa J. Toita M. Yoshida M. J. Virol. 1989; 63: 3234-3239Crossref PubMed Google Scholar, 6Giam C.Z. Xu Y.L. J. Biol. Chem. 1989; 264: 15236-15241Abstract Full Text PDF PubMed Google Scholar). Each of the viral 21-bp-repeats contains a cAMP responseelement (CRE) core flanked by 5′ G-rich and 3′ C-rich sequences. The 21-bp repeats, in collaboration with the cellular transcription factors, cAMP responseelement binding protein (CREB), CREB/activating transcriptionfactor 1 (ATF-1) heterodimer, and to a lesser extent, ATF-1 homodimer, form nucleoprotein complexes uniquely capable of recruiting Tax into ternary complexes that mediate trans-activation (7Zhao L.J. Giam C.Z. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11445-11449Crossref PubMed Scopus (168) Google Scholar, 8Zhao L.J. Giam C.Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7070-7074Crossref PubMed Scopus (296) Google Scholar, 9Suzuki T. Fujisawa J.I. Toita M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 610-614Crossref PubMed Scopus (244) Google Scholar, 10Goren I. Semmes O.J. Jeang K.T. Moelling K. J. Virol. 1995; 69: 5806-5811Crossref PubMed Google Scholar, 11Bantignies F. Rousset R. Desbois C. Jalinot P. Mol. Cell. Biol. 1996; 16: 2174-2182Crossref PubMed Scopus (53) Google Scholar, 12Shnyreva M. Munder T. J. Virol. 1996; 70: 7478-7484Crossref PubMed Google Scholar). Whereas CREB binds to the CRE irrespective of DNA sequence context, the stable assembly of Tax, CREB, and DNA into a ternary complex and Tax-mediated trans-activation require the CRE and the 5′ G-rich and 3′ C-rich flanking sequences present in the viral 21-bp repeats (11Bantignies F. Rousset R. Desbois C. Jalinot P. Mol. Cell. Biol. 1996; 16: 2174-2182Crossref PubMed Scopus (53) Google Scholar, 12Shnyreva M. Munder T. J. Virol. 1996; 70: 7478-7484Crossref PubMed Google Scholar, 13Derse D. J. Virol. 1987; 61: 2462-2471Crossref PubMed Google Scholar, 14Montagne J. Beraud C. Crenon I. Lombard Platet G. Gazzolo L. Sergeant A. Jalinot P. EMBO J. 1990; 9: 957-964Crossref PubMed Scopus (55) Google Scholar, 15Paca Uccaralertkun S. Zhao L.J. Adya N. Cross J.V. Cullen B.R. Boros I.M. Giam C.Z. Mol. Cell. Biol. 1994; 14: 456-462Crossref PubMed Google Scholar, 16Yin M.J. Gaynor R.B. Mol. Cell. Biol. 1996; 16: 3156-3168Crossref PubMed Scopus (83) Google Scholar, 17Yin M.J. Paulssen E. Seeler J. Gaynor R.B. J. Virol. 1995; 69: 6209-6218Crossref PubMed Google Scholar, 18Boros I.M. Tie F. Giam C.Z. Virology. 1995; 214: 207-214Crossref PubMed Scopus (17) Google Scholar).In vitro selection also indicates that DNA sequences bound preferentially by the Tax/CREB complex contain the CRE motif flanked by exceedingly long stretches of 5′ G-rich or 3′ C-rich sequences and bear striking resemblance to the HTLV-I 21-bp repeats (15Paca Uccaralertkun S. Zhao L.J. Adya N. Cross J.V. Cullen B.R. Boros I.M. Giam C.Z. Mol. Cell. Biol. 1994; 14: 456-462Crossref PubMed Google Scholar). These results demonstrate directly that HTLV-I Tax is evolved principally toward activating transcription from the viral cis regulatory element, the HTLV-I 21-bp repeats. The molecular basis for the DNA sequence specificity of Tax trans-activation is not well understood. DNase I footprinting and methylation interference performed on the Tax·CREB·21-bp-repeat complex revealed no protein protection of the G-rich and C-rich sequences (15Paca Uccaralertkun S. Zhao L.J. Adya N. Cross J.V. Cullen B.R. Boros I.M. Giam C.Z. Mol. Cell. Biol. 1994; 14: 456-462Crossref PubMed Google Scholar). Further, naturally occurring or genetically engineered single nucleotide substitutions in the G-rich and C-rich sequences do not affect their function (6Giam C.Z. Xu Y.L. J. Biol. Chem. 1989; 264: 15236-15241Abstract Full Text PDF PubMed Google Scholar). Interestingly, recent data indicate that Tax may be involved in contacting the minor groove of the G/C-rich sequences (19Lenzmeier B.A. Giebler H.A. Nyborg J.K. Mol. Cell. Biol. 1998; 18: 721-731Crossref PubMed Google Scholar). The specificity for the flanking sequences requires a specific interaction between Tax and the basic region of CREB (20Adya N. Zhao L.J. Huang W. Boros I. Giam C.Z. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5642-5646Crossref PubMed Scopus (83) Google Scholar). Tax interacts with CREB via the latter's basic domain and amino acid residues in its immediate vicinity (11Bantignies F. Rousset R. Desbois C. Jalinot P. Mol. Cell. Biol. 1996; 16: 2174-2182Crossref PubMed Scopus (53) Google Scholar, 12Shnyreva M. Munder T. J. Virol. 1996; 70: 7478-7484Crossref PubMed Google Scholar, 20Adya N. Zhao L.J. Huang W. Boros I. Giam C.Z. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5642-5646Crossref PubMed Scopus (83) Google Scholar, 21Yin M.J. Gaynor R.B. J. Mol. Biol. 1996; 264: 20-31Crossref PubMed Scopus (47) Google Scholar, 22Perini G. Wagner S. Green M.R. Nature. 1995; 376: 602-605Crossref PubMed Scopus (135) Google Scholar). Like CREB, Tax functions as a dimer (22Perini G. Wagner S. Green M.R. Nature. 1995; 376: 602-605Crossref PubMed Scopus (135) Google Scholar, 23Tie F. Adya N. Greene W.C. Giam C.Z. J. Virol. 1996; 70: 8368-8374Crossref PubMed Google Scholar). Analyses of Tax mutants indicate that the CREB-binding domain is located in the NH2 terminus, while the subunit dimerization domain resides in the middle section of Tax (10Goren I. Semmes O.J. Jeang K.T. Moelling K. J. Virol. 1995; 69: 5806-5811Crossref PubMed Google Scholar, 23Tie F. Adya N. Greene W.C. Giam C.Z. J. Virol. 1996; 70: 8368-8374Crossref PubMed Google Scholar, 24Adya N. Giam C.Z. J. Virol. 1995; 69: 1834-1841Crossref PubMed Google Scholar). CREB and ATFs are prototypic bZip proteins noted for the common basic domain-leucine zipper structure responsible for sequence specific DNA recognition and intersubunit protein-protein interaction, respectively (25Hai T.W. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (753) Google Scholar). Many members of the bZip family, including the mammalian CREB, ATF, c-Jun/c-Fos, and yeast GCN4, bind either the 7-bp AP-1 motif (TGA(C/G)TCA) or the related CRE motif (TGACGTCA). Remarkably, a third group of the families whose members include NF-IL6 (also known as C/EBP-β) and C/EBP, bind a set of DNA elements (T(T/G)NNGNAA(T/G) and CCAAT box, respectively) whose sequences differ significantly from the AP-1 site and CRE. CREB becomes phosphorylated and activated as a function of the cAMP- or Ca2+-mediated signaling process (26Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2041) Google Scholar, 27Gonzalez G.A. Yamamoto K.K. Fischer W.H. Karr D. Menzel P. Biggs W. Vale W.W. Montminy M.R. Nature. 1989; 337: 749-752Crossref PubMed Scopus (644) Google Scholar, 28Sun P. Lou L. Maurer R.A. J. Biol. Chem. 1996; 271: 3066-3073Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Both protein kinase A and Ca2+/calmodulin-dependent kinases (I and IV) have been shown to phosphorylate CREB at Ser133to activate CRE/CREB-mediated transcription (26Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2041) Google Scholar, 27Gonzalez G.A. Yamamoto K.K. Fischer W.H. Karr D. Menzel P. Biggs W. Vale W.W. Montminy M.R. Nature. 1989; 337: 749-752Crossref PubMed Scopus (644) Google Scholar, 28Sun P. Lou L. Maurer R.A. J. Biol. Chem. 1996; 271: 3066-3073Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). A 265-kDa protein called CBP (CREB binding protein), and its cellular homologue, p300, specifically bind the Ser133-phosphorylated form of CREB (29Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1758) Google Scholar) and function as transcriptional co-activators of CREB (30Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1279) Google Scholar). Kwoket al. (31Kwok R.P. Laurance M.E. Lundblad J.R. Goldman P.S. Shih H. Connor L.M. Marriott S.J. Goodman R.H. Nature. 1996; 380: 642-646Crossref PubMed Scopus (308) Google Scholar) have shown recently that Tax interacts directly with the CBP and p300. These data have recently been confirmed and extended (32Giebler H.A. Loring J.E. van Orden K. Colgin M.A. Garrus J.E. Escudero K.W. Brauweiler A. Nyborg J.K. (2) Mol. Cell. Biol. 1997; 17: 5156-5164Crossref PubMed Scopus (164) Google Scholar). Indeed, in in vitro protein/DNA binding reactions, the inclusion of CBP/p300 results in a highly stable quaternary nucleoprotein complex containing the 21-bp repeat/CREB/Tax/CBP-p300 (see “Results” and Ref. 32Giebler H.A. Loring J.E. van Orden K. Colgin M.A. Garrus J.E. Escudero K.W. Brauweiler A. Nyborg J.K. (2) Mol. Cell. Biol. 1997; 17: 5156-5164Crossref PubMed Scopus (164) Google Scholar). 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Cell. 1992; 71: 1223-1237Abstract Full Text PDF PubMed Scopus (813) Google Scholar, 34Konig P. Richmond T.J. J. Mol. Biol. 1993; 233: 139-154Crossref PubMed Scopus (259) Google Scholar, 35Glover J.N. Harrison S.C. Nature. 1995; 373: 257-261Crossref PubMed Scopus (663) Google Scholar, 36Keller W. Konig P. Richmond T.J. J. Mol. Biol. 1995; 254: 657-667Crossref PubMed Scopus (162) Google Scholar). In this study, we show that an α-helix extending beyond the DNA-binding domain of CREB bZip is required for Tax binding. Along this extended helix, three amino acid (aa) residues, Arg284, Met291, and Glu299, located on the opposing side of the invariant DNA recognition residues form the contact surface for Tax. The Tax-contact residues span a distance of approximately 23 Å along most of the length of the basic domain and beyond. Interestingly, these three amino acid residues point away from the major groove of the DNA and are posed adjacent to the minor groove of the G/C-rich sequences flanking the CRE motif, which we and others have shown to be critical for Tax binding (11Bantignies F. Rousset R. Desbois C. Jalinot P. Mol. Cell. Biol. 1996; 16: 2174-2182Crossref PubMed Scopus (53) Google Scholar, 12Shnyreva M. Munder T. J. Virol. 1996; 70: 7478-7484Crossref PubMed Google Scholar, 15Paca Uccaralertkun S. Zhao L.J. Adya N. Cross J.V. Cullen B.R. Boros I.M. Giam C.Z. Mol. Cell. Biol. 1994; 14: 456-462Crossref PubMed Google Scholar, 19Lenzmeier B.A. Giebler H.A. Nyborg J.K. Mol. Cell. Biol. 1998; 18: 721-731Crossref PubMed Google Scholar). Our data lend further support to the notion that Tax is involved in minor groove DNA contact with the G/C-rich flanking sequences after its binding to CREB. We thank Dr. R. Goodman for the gift of the GST-CBP451–682 construct.

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