title: T-loop phosphorylated Cdk9 localizes to nuclear speckle domains which may serve as sites of active P-TEFb function and exchange between the Brd4 and 7SK/HEXIM1 regulatory complexes.
abstract: P-TEFb functions to induce the elongation step of RNA polymerase II transcription by phosphorylating the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Core P-TEFb is comprised of Cdk9 and a cyclin regulatory subunit, with Cyclin T1 being the predominant Cdk9-associated cyclin. The kinase activity of P-TEFb is dependent on phosphorylation of the Thr186 residue located within the T-loop domain of the Cdk9 subunit. Here, we used immunofluorescence deconvolution microscopy to examine the subcellular distribution of phospho-Thr186 Cdk9/Cyclin T1 P-TEFb heterodimers. We found that phospho-Thr186 Cdk9 displays a punctate distribution throughout the non-nucleolar nucleoplasm and it co-localizes with Cyclin T1 almost exclusively within nuclear speckle domains. Phospho-Thr186 Cdk9 predominantly co-localized with the hyperphosphorylated forms of RNA polymerase II. Transient expression of kinase-defective Cdk9 mutants revealed that neither is Thr186 phosphorylation or kinase activity required for Cdk9 speckle localization. Lastly, both the Brd4 and HEXIM1 proteins interact with P-TEFb at or very near speckle domains and treatment of cells with the Cdk9 inhibitor flavopiridol alters this distribution. These results indicate that the active form of P-TEFb resides in nuclear speckles and raises the possibility that speckles are sites of P-TEFb function and exchange between negative and positive P-TEFb regulatory complexes.
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
authors.forename: Eugene C
authors.initials: EC
authors.lastname: Dow
authors.forename: Hongbing
authors.initials: H
authors.lastname: Liu
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Rice
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology*; D056906:Cyclin T / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D055503:Protein Multimerization; D021381:Protein Transport; D012319:RNA Polymerase II / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D013912:Threonine; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D000092468:Bromodomain Containing Proteins
chemical_list: D018797:Cell Cycle Proteins; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9; D005419:Flavonoids; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D010880:Piperidines; D042925:Positive Transcriptional Elongation Factor B; D047428:Protein Kinase Inhibitors; D012319:RNA Polymerase II; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017411:Ribonucleoproteins, Small Nuclear; D013912:Threonine; D014157:Transcription Factors; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C503684:CCNT1 protein, human; C476123:HEXIM1 protein, human; C077990:alvocidib; C467969:CDK9 protein, human