HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 26, 24968-24977, July 1, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/26/24968    most recent M501431200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schulte, A. Articles by Geyer, M. Search for Related Content PubMed PubMed Citation Articles by Schulte, A. Articles by Geyer, M. Social Bookmarking                      What's this?

Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat^*

Antje Schulte, Nadine Czudnochowski, Matjaz Barboric¶, André Schönichen, Dalibor Blazek, B. Matija Peterlin, and Matthias Geyer||

From the Max-Planck-Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany and the Departments of Medicine, Microbiology, and Immunology, Rosalind Russell Medical Research Center, University of California at San Francisco, San Francisco, California 94143-0703

The active form of the positive transcription elongation factor b (P-TEFb) consists of cyclin T and the kinase Cdk9. P-TEFb stimulates transcription by phosphorylating the C-terminal domain of RNA polymerase II. It becomes inactivated when associated in a tetrameric complex with the abundant 7SK small nuclear RNA and the recently identified protein Hexim1. In this study, we identified a stable and soluble C-terminal domain (residues 255–359) in Hexim1 of 12.5-kDa size that binds the cyclin boxes of Cyclin T1. Functional assays in HeLa cells showed that this cyclin T-binding domain (TBD) is required for the binding of Hexim1 to P-TEFb and inhibition of transcriptional activity in vivo. Analytical gel filtration and GST pull-down experiments revealed that both full-length Hexim1 and the TBD are homodimers. Isothermal titration calorimetry yielded a weak multimer for the TBD with a multimerization constant of 1.3 x 10³ M. The binding affinity between the TBD and cyclin T1 was analyzed with fluorescence spectroscopy methods, using a dansyl-based fluorescence label at position G257C. Equilibrium fluorescence titration and stopped flow fast kinetics yield a dissociation constant of 1.2 µM. Finally, we tested the effect of the HIV-1 Tat protein on the cyclin T1-TBD complex formation. GST pull-down experiments and size exclusion chromatography exhibit a mutually exclusive binding of the two effectors to cyclin T1. Our data suggest a model where HIV-1 Tat competes with Hexim1 for cyclin T1 binding, thus releasing P-TEFb from the inactive complex to stimulate the transcription of HIV-1 gene expression.

Received for publication, February 7, 2005 , and in revised form, April 20, 2005.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant GE-976/2 (to M. G.) and by National Institutes of Health Grant RO1 AI49104 (to B. M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported in part by American Foundation for AIDS Research Grant 106584-36-RFNT.

^|| to whom correspondence should be addressed: Max Planck Institute for Molecular Physiology, Dept. Physical Biochemistry, Otto-Hahn-Str. 11, Dortmund D-44227, Germany. Tel.: 49-231-133-2366; Fax: 49-231-133-2399; E-mail: matthias.geyer{at}mpi-dortmund.mpg.de.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				N. Shimizu, N. Yoshikawa, T. Wada, H. Handa, M. Sano, K. Fukuda, M. Suematsu, T. Sawai, C. Morimoto, and H. Tanaka Tissue- and Context-Dependent Modulation of Hormonal Sensitivity of Glucocorticoid-Responsive Genes by Hexamethylene Bisacetamide-Inducible Protein 1 Mol. Endocrinol., December 1, 2008; 22(12): 2609 - 2623. [Abstract] [Full Text] [PDF]

				J. K. Jadlowsky, M. Nojima, T. Okamoto, and K. Fujinaga Dominant negative mutant cyclin T1 proteins that inhibit HIV transcription by forming a kinase inactive complex with Tat J. Gen. Virol., November 1, 2008; 89(11): 2783 - 2787. [Abstract] [Full Text] [PDF]

				C. Barrandon, F. Bonnet, V. T. Nguyen, V. Labas, and O. Bensaude The Transcription-Dependent Dissociation of P-TEFb-HEXIM1-7SK RNA Relies upon Formation of hnRNP-7SK RNA Complexes Mol. Cell. Biol., October 15, 2007; 27(20): 6996 - 7006. [Abstract] [Full Text] [PDF]

				S. A. Dames, A. Schonichen, A. Schulte, M. Barboric, B. M. Peterlin, S. Grzesiek, and M. Geyer Structure of the Cyclin T binding domain of Hexim1 and molecular basis for its recognition of P-TEFb PNAS, September 4, 2007; 104(36): 14312 - 14317. [Abstract] [Full Text] [PDF]

				S. C. Sedore, S. A. Byers, S. Biglione, J. P. Price, W. J. Maury, and D. H. Price Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR Nucleic Acids Res., July 26, 2007; 35(13): 4347 - 4358. [Abstract] [Full Text] [PDF]

				M. Barboric, J. H. N. Yik, N. Czudnochowski, Z. Yang, R. Chen, X. Contreras, M. Geyer, B. Matija Peterlin, and Q. Zhou Tat competes with HEXIM1 to increase the active pool of P-TEFb for HIV-1 transcription Nucleic Acids Res., March 19, 2007; 35(6): 2003 - 2012. [Abstract] [Full Text] [PDF]

				J. Kohoutek, D. Blazek, and B. M. Peterlin Hexim1 sequesters positive transcription elongation factor b from the class II transactivator on MHC class II promoters PNAS, November 14, 2006; 103(46): 17349 - 17354. [Abstract] [Full Text] [PDF]

				Q. Zhou and J. H. N. Yik The Yin and Yang of P-TEFb Regulation: Implications for Human Immunodeficiency Virus Gene Expression and Global Control of Cell Growth and Differentiation Microbiol. Mol. Biol. Rev., September 1, 2006; 70(3): 646 - 659. [Abstract] [Full Text] [PDF]

				S. Shojania and J. D. O'Neil HIV-1 Tat Is a Natively Unfolded Protein: THE SOLUTION CONFORMATION AND DYNAMICS OF REDUCED HIV-1 Tat-(1-72) BY NMR SPECTROSCOPY J. Biol. Chem., March 31, 2006; 281(13): 8347 - 8356. [Abstract] [Full Text] [PDF]

				A. Schonichen, M. Alexander, J. E. Gasteier, F. E. Cuesta, O. T. Fackler, and M. Geyer Biochemical Characterization of the Diaphanous Autoregulatory Interaction in the Formin Homology Protein FHOD1 J. Biol. Chem., February 24, 2006; 281(8): 5084 - 5093. [Abstract] [Full Text] [PDF]

				D. Blazek, M. Barboric, J. Kohoutek, I. Oven, and B. M. Peterlin Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb Nucleic Acids Res., December 23, 2005; 33(22): 7000 - 7010. [Abstract] [Full Text] [PDF]

				C. Dulac, A. A. Michels, A. Fraldi, F. Bonnet, V. T. Nguyen, G. Napolitano, L. Lania, and O. Bensaude Transcription-dependent Association of Multiple Positive Transcription Elongation Factor Units to a HEXIM Multimer J. Biol. Chem., August 26, 2005; 280(34): 30619 - 30629. [Abstract] [Full Text] [PDF]