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Analysis of the large inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated at threonine 186
Journal article   Open access   Peer reviewed

Analysis of the large inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated at threonine 186

Qintong Li, Jason P Price, Sarah A Byers, Dongmei Cheng, Junmin Peng and David H Price
The Journal of biological chemistry, Vol.280(31), pp.28819-28826
08/05/2005
DOI: 10.1074/jbc.M502712200
PMID: 15965233
url
https://doi.org/10.1074/jbc.M502712200View
Published (Version of record) Open Access

Abstract

Positive transcription elongation factor b (P-TEFb) regulates eukaryotic gene expression at the level of elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein, HEXIM1 or HEXIM2. To further understand how P-TEFb is regulated, we analyzed the stoichiometry of all the known components of the large, inactive P-TEFb complex. Mutational analyses of a putative coiled coil region in the carboxyl-terminal portion of HEXIM1 revealed that the protein is a dimer in solution and remains a dimer after binding to 7SK. Although a HEXIM1 dimer contains two potential RNA binding motifs and ultimately recruits two P-TEFb molecules, it associates with only one molecule of RNA. The first 172 nucleotides of the 330-nucleotide 7SK are sufficient to bind HEXIM1 or HEXIM2, and then recruit and inhibit P-TEFb. Deletion of the first 121 amino acids of HEXIM1 allowed it to inhibit P-TEFb partially in the absence of 7SK RNA. Mutation of a conserved tyrosine (Tyr(271) in HEXIM1) to alanine or glutamate or mutation of a conserved phenylalanine (Phe(208)) to alanine, aspartate, or lysine, resulted in loss of inhibition of P-TEFb, but did not affect formation of the 7SK.HEXIM.P-TEFb complex. Analysis of T-loop phosphorylation in Cdk9 indicated that phosphorylation of Thr(186), but not Ser(175), was essential for kinase activity and for recruitment of P-TEFb to the 7SK.HEXIM complex. A model illustrates what is currently known about how HEXIM proteins, 7SK, and P-TEFb assemble to maintain an activated kinase in a readily available, but inactive form.
 Amino Acid Sequence Phosphorylation Threonine CDC2-CDC28 Kinases - chemistry Humans RNA-Binding Proteins - chemistry Positive Transcriptional Elongation Factor B - chemistry CDC2-CDC28 Kinases - metabolism Models, Molecular Positive Transcriptional Elongation Factor B - metabolism Cyclin-Dependent Kinase 2 Protein Conformation Ribonucleoproteins, Small Nuclear - chemistry HeLa Cells Dimerization Macromolecular Substances - metabolism RNA-Binding Proteins - metabolism Ribonucleoproteins, Small Nuclear - metabolism

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