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. 1999 Jul;73(7):5448-58.
doi: 10.1128/JVI.73.7.5448-5458.1999.

Human and rodent transcription elongation factor P-TEFb: interactions with human immunodeficiency virus type 1 tat and carboxy-terminal domain substrate

Y Ramanathan  1 , S M Reza, T M Young, M B Mathews, T Pe'ery
Affiliations

Human and rodent transcription elongation factor P-TEFb: interactions with human immunodeficiency virus type 1 tat and carboxy-terminal domain substrate

Y Ramanathan et al. J Virol. 1999 Jul.

Abstract

The human immunodeficiency virus type 1 transcriptional regulator Tat increases the efficiency of elongation, and complexes containing the cellular kinase CDK9 have been implicated in this process. CDK9 is part of the Tat-associated kinase TAK and of the elongation factor P-TEFb (positive transcription elongation factor-b), which consists minimally of CDK9 and cyclin T. TAK and P-TEFb are both able to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II, but their relationships to one another and to the stimulation of elongation by Tat are not well characterized. Here we demonstrate that human cyclin T1 (but not cyclin T2) interacts with the activation domain of Tat and is a component of TAK as well as of P-TEFb. Rodent (mouse and Chinese hamster) cyclin T1 is defective in Tat binding and transactivation, but hamster CDK9 interacts with human cyclin T1 to give active TAK in hybrid cells containing human chromosome 12. Although TAK is phosphorylated on both serine and threonine residues, it specifically phosphorylates serine 5 in the CTD heptamer. TAK is found in the nuclear and cytoplasmic fractions of human cells as a large complex (approximately 950 kDa). Magnesium or zinc ions are required for the association of Tat with the kinase. We suggest a model in which Tat first interacts with P-TEFb to form the TAK complex that engages with TAR RNA and the elongating transcription complex, resulting in hyperphosphorylation of the CTD on serine 5 residues.

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Figures

FIG. 1
FIG. 1
Specificity of CTD phosphorylation by TAK/P-TEFb. (A) Detection of TAK activity with CTD3 and RNA pol II substrates. The indicated GST-Tat or GST-Tat mutant fusion proteins (lanes 1 to 4, 7, and 8) were used to isolate TAK from cytoplasmic fractions of 293 cells by the GST pull-down procedure. For comparison, IP-kinase assays were conducted with immunoprecipitates prepared with anti-CDK9 or control antibodies (lanes 5 and 6, respectively). Kinase reaction mixtures contained 13.5 μg of CTD3 (lanes 1 to 6) or 20 ng of purified Drosophila RNA pol II (lanes 7 and 8) as the substrate. (B) CTD3 phosphorylation by TAK eluted from immunoprecipitated CDK9 complexes. The immunoprecipitates were washed extensively, and complexes were eluted for 18 h with the indicated amounts of CDK9 C-terminal peptide. The eluate was subjected to TAK assay, and the supernatant was examined by gel electrophoresis and autoradiography. The control was the standard TAK assay with 293 cell S100 and GST-Tat48Δ. (C) Detection of CDK9 in the eluates after binding to GST-Tat48Δ beads. The proteins bound to the beads in panel B were examined by Western blotting with anti-CDK9 antibody.
FIG. 2
FIG. 2
TAK in CHO-human hybrid cell extracts. (A) CTD3 phosphorylation by TAK pulled down from different cell extracts: human (293, HeLa, and GM07890), monkey (COS), rodent (3T3 and CHO), and the rodent-human hybrids CHO(6) and CHO(12). (B) Western blotting of proteins bound to GST-Tat48Δ beads from the indicated cell extracts by using anti-CDK9 and anti-cyclin T1 antibodies. (C) Western blot analysis of proteins in unfractionated cell extracts with the same antibodies as in panel B. (D) IP-kinase assays conducted with the same cell extracts with CTD3 as the substrate.
FIG. 3
FIG. 3
Phosphoamino acid analysis of CTD3 peptide and CDK9. CTD3, phosphorylated in TAK assays, resolved into two bands upon gel electrophoresis. The upper band (A) and lower band (B) were analyzed separately. Positions of phosphoamino acid markers are circled. CDK9 was phosphorylated in IP-kinase assays (C) or TAK assays (D) and examined in the same way.
FIG. 4
FIG. 4
Selectivity of CTD3 phosphorylation by CDK9 and CDK7. (A) Sequence of wild-type (WT) CTD3 and mutant peptides. Substitutions are denoted by the underlined boldface letters. (B) Phosphorylation of the indicated CTD3 peptides in TAK assays. (C) Comparison of CTD3 phosphorylation by CDK9 and CDK7 complexes. CDK9 was monitored in TAK assays conducted with various amounts of 293 cell S100 (15 μg/ml). CDK7 was monitored by using Sf9 cell extracts (∼60 μg) containing either recombinant CAK or recombinant CDK7 and cyclin H. The control was the kinase assay with mock-infected Sf9 cell extract.
FIG. 5
FIG. 5
Kinetics of TAK activity and nucleotide usage by TAK. (A) Time course of CTD3 phosphorylation by TAK from 293 cell S100. Reactions were stopped at the indicated time points. (B) Kinase reaction mixtures containing [γ-32P]ATP were supplemented with the indicated unlabeled nucleotides at the concentrations shown. Control, no unlabeled nucleotides added.
FIG. 6
FIG. 6
Effects of Mg and EDTA on TAK activity. (A) 293 cell S100 fractions were treated with EDTA and MgCl2 at the final concentrations indicated and then subjected to TAK assay. After the kinase assay, the supernatant was examined for CTD3 phosphorylation. (B) Western blot analysis with anti-CDK9 antibody of the proteins bound to GST-Tat48Δ beads in the same experiment as for panel A. (C) Western blot analysis with anti-CDK9 antibody of proteins remaining in the supernatant after binding to GST-Tat48Δ beads in the same experiment as for panels A and B. Load, sample of 293 cell S100. (D) IP-kinase assays conducted with 293 cell S100 (15 mg/ml) subjected to treatment with EDTA and Mg2+ as indicated.
FIG. 7
FIG. 7
Separation of TAK complexes by gel filtration and sedimentation. (A) CTD3 phosphorylation by TAK pulled down from fractions after resolution of 293 cell S100 in a gel filtration column. Molecular mass markers (in kilodaltons) are indicated by arrows. Triangles show the estimated molecular masses of the large complex (950 kDa), CDK9/cyclin T complex (130 kDa), cyclin T (90 kDa), and CDK9 (40 kDa). (B and C) The same fractions were examined by Western blotting with antibodies to CDK9 (B) and cyclin T1 (C). Load, sample of the 293 cell S100 loaded on the column. (D) Cytoplasmic extracts from 293 cells in 150 mM KCl or adjusted to 1 M KCl were fractionated in glycerol gradients. Fractions were subjected to TAK assays, and the phosphorylated CTD3 bands were quantified, normalized, and plotted.
FIG. 8
FIG. 8
Schematic representation of Tat delivery to its nuclear transactivation site. The model depicts TAK assembly in the cytoplasm. P-TEFb, composed of CDK9, cyclin T1, and other components, interacts with the activation domain (AD) of Tat. In the nucleus, the TAK complex (P-TEFb–Tat) interacts with the transcription complex via cyclin T1 and the basic domain (BD) of Tat, both of which bind to TAR. Through phosphorylation of the pol II CTD, the elongating complex becomes more processive and Tat transactivation is achieved. (See text for details.)
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