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

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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.

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