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. J. Cell. Physiol. 224:84–93, 2010 © 2010 Wiley‐Liss, Inc.