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Molecular and Cellular Biology, September 2000, p. 6958-6969, Vol. 20, No. 18
Regulatory Biology
Laboratory1 and Molecular Biology and
Virology Laboratory,2 The Salk Institute for
Biological Studies, La Jolla, California 92037, and McArdle
Laboratory for Cancer Research, University of Wisconsin, Madison,
Wisconsin 537063
Received 7 February 2000/Returned for modification 31 March
2000/Accepted 19 June 2000
Human immunodeficiency virus type 1 (HIV-1) Tat interacts with
cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds
cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote
transcription elongation. We show here that Tat also stimulates
phosphorylation of affinity-purified core RNA
polymerase II and glutathione
S-transferase-C-terminal-domain substrates by CycT1-CDK9,
but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant
Tat-P-TEFb complexes with ATP enhanced binding to TAR RNA
dramatically, and the C-terminal half of CycT1 masked binding of Tat to
TAR RNA in the absence of ATP. ATP incubation lead to
autophosphorylation of CDK9 at multiple C-terminal
Ser and Thr residues, and full-length CycT1 (amino acids 728)
[CycT1(1-728)], but not truncated CycT1(1-303), was also
phosphorylated by CDK9. P-TEFb complexes containing a catalytically
inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of
ATP, as did a C-terminal truncated CDK9 mutant that was catalytically
active but unable to undergo autophosphorylation.
Analysis of different Tat proteins revealed that the 101-amino-acid SF2
HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of
phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked
binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon
autophosphorylation. Replacement of CDK9
phosphorylation sites with negatively charged residues
restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more
potent inhibitor of transcription in vitro. Taken together, these
results demonstrate that CDK9 phosphorylation is
required for high-affinity binding of Tat-P-TEFb to TAR RNA and that
the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
CDK9 Autophosphorylation Regulates High-Affinity Binding of
the Human Immunodeficiency Virus Type 1 Tat-P-TEFb Complex to
TAR RNA
*
Corresponding author. Mailing address: Regulatory
Biology Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 452-1122. Fax: (858)
535-8194. E-mail: jones{at}salk.edu.
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