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, 224 (1), 84-93

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

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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

Eugene C Dow et al. J Cell Physiol.

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.

Figures

Fig. 1
Fig. 1
The p-Cdk9 antibody is specific for T-loop activated Cdk9. Indirect immunofluorescence was conducted on HeLa cells to evaluate the specificity and staining pattern of the phospho-T186 antiserum (p-Cdk9). A: (upper parts) Cells were labeled using a 1:50 dilution of the p-Cdk9 antibody (determined as the optimum dilution by titration, data not shown) which was detected with an Alexa-488 (green) conjugated secondary antibody and the DNA was counterstained with DAPI. A: (lower parts) HeLa cells were labeled with a working dilution (1:50) of the p-Cdk9 antibody pre-incubated with a 10-fold molar excess of the peptide used to generate it. As can be seen, the cognate peptide effectively competes out the p-Cdk9 signal, demonstrating the specificity of this antiserum for use in indirect immunofluorescence. B: A comparison of the subnuclear distribution of T-loop activated Cdk9 (p-Cdk9) versus total Cdk9 (pan-Cdk9) shows that a more defined punctate, non-nucleolar staining pattern is seen with the p-Cdk9 signal with large clusters reminiscent of nuclear speckles. Bar, 10 µm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 2
Fig. 2
The active form of P-TEFb is located within nuclear speckles. HeLa cells were triple immunolabeled for SC35 (blue), p-Cdk9 (green), and Cyclin T1 (red) as shown in the upper parts. Co-localization of p-Cdk9 with SC35 (aqua), Cyclin T1 with SC35 (purple), and p-Cdk9 with Cyclin T1 (yellow) are shown in the middle parts. As can be seen, the large clusters of p-Cdk9 co-localize with nuclear speckles and to a high degree with Cyclin T1 (middle parts). The merge of all three channels shows that almost all of the active form of P-TEFb (p-Cdk9/Cyclin T1) is found within nuclear speckles. In the merge part areas of co-localization between p-Cdk9 and Cyclin T1 with nuclear speckles (SC35) appear as white, whereas those outside the speckle domains are indicated by yellow. Bar, 10 µm.
Fig. 3
Fig. 3
T-loop activated Cdk9 co-localizes mostly with the hyperphosphorylated forms of RNAPII. HeLa cells were dual immunolabeled for p-Cdk9 (green) and the various phosphoforms of RNAPII (red). Co-localization between p-Cdk9 and hypophosphorylated RNAPII (8WG16), Ser5-phosphorylated RNAPII (H14), and Ser2-phosphorylated RNAPII (H5) is shown in the upper, middle, and lower merge parts, respectively. Areas of co-localization are indicated by the yellow color in the merge parts and an enlargement of a speckle-like cluster of p-Cdk9 (boxed region in merge) from each cell is shown in the insets. Bar, 10 µm.
Fig. 4
Fig. 4
Cdk9 does not require Thr186 phosphorylation or kinaseactivity for nuclear speckle localization. HeLa cells grown on glass coverslips were transfected with 500 ng of either: pBABE-Flag-Cdk9T186A-IRES-eGFP (fCdk9-T186A, upper parts), pBABE-Flag-Cdk9-IRES-eGFP (fCdk9-WT, middle parts), or pBABE-Flag-Cdk9D167N-IRES-eGFP (fCdk9-D167N, bottom parts) and fixed at 24 h post-transfection. The fixed cells were then immunolabeled for Flag (red) and SC35 (blue). The eGFP (green) was used as an internal control in conjunction with the Flag signal for assessing cells with similar expression levels. Immunoblots were also conducted to confirm that the expression levels of eGFP and the epitope-tagged Cdk9 proteins were similar in all cultures examined (data not shown). As can be seen, all three of the Flag-tagged Cdk9 proteins co-localize with SC35 (purple) to a similar extent, and show a similar non-nucleolar distribution throughout the nucleoplasm. An enlargement of a nuclear speckle from each of the three cells (boxed region on SC35/Flag parts) is shown on the right. Bar, 10 µm.
Fig. 5
Fig. 5
Co-localization of the small and large P-TEFb complexes with nuclear speckles. A: HeLa cells were triple immunolabeled for SC35 (blue), Cyclin T1 (red), and HEXIM1 or Brd4 (green) as shown in parts 1–3 and 7–9, respectively. The co-localization of Cyclin T1 with SC35 (purple), HEXIM1 with SC35 (aqua), and Cyclin T1 with HEXIM1 (yellow) are shown in parts 4–6. The areas of co-localization between Cyclin T1 and SC35 (purple), Brd4 and SC35 (aqua), and Cyclin T1 with Brd4 (yellow) are shown in parts 10–12. As can be seen, both the HEXIM1 and Brd4 proteins co-localize with nuclear speckles (parts 5 and 11) and both HEXIM1 and Brd4 co-localize with Cyclin T1 (parts 6 and 12). B: Merge parts from the cells in A, in which co-localization of the large complex (HEXIM1/Cyclin T1) with nuclear speckles (SC35) is indicated by white and shown in the upper part, and co-localization of the small complex (Brd4/Cyclin T1) with nuclear speckles (SC35) is shown in the bottom part (also indicated by white). Areas of localization of the large and small complexes outside of the speckles regions are indicated by yellow in these parts. As can be seen, while both the large and small P-TEFb complexes co-localize with nuclear speckles (white), the small complex has a very tight co-localization pattern within the speckles, whereas the large complex localizes not only in the speckles, but also at many areas surrounding the outside of these regions (yellow). Bar, 10 µm.
Fig. 6
Fig. 6
Effects of flavopiridol on the localization of active P-TEFb and the small and large P-TEFb complexes. A: HeLa cells were treated with 500 nM flavopiridol for 2 h prior to immunofluorescence labeling for SC35 (blue), Cyclin T1 (red), and either Brd4 (green, upper parts), p-Cdk9 (green, middle parts), or HEXIM1 (green, bottom parts). As can be seen, flavopiridol causes the speckles to round up (parts 1, 8, and 15) and Cyclin T1 to co-localizegreatly with the nuclearspeckles (indicated by purple in parts4, 11, and 18). Similarly, both Brd4 and p-Cdk9 also greatly co-localize with speckles (indicated by aqua in parts 5 and 12) and with Cyclin T1 (indicated by yellow in parts 6 and 13). In contrast, flavopiridol causes HEXIM1 to become more diffusely scattered throughout the nucleoplasm (part 17) and abatement of its co-localization with speckles (part 19) and Cyclin T1 (part 20). A merge part for each cell specifies areas of co-localization between Brd4/Cyclin T1, p-Cdk9/Cyclin T1, and HEXIM1/Cyclin T1 complexes with nuclear speckles (indicated by white in parts 7, 14, and 21, respectively) and outside of speckles (indicated by yellow in parts 7, 14, and 21 respectively). Bar, 10 µm. B: HeLa cells were treated with 500 nM flavopiridol or DMSO (solvent control) for 2 h prior to preparation of cell lysates. The expression levels of the indicated proteins were examined in immunoblots. No significant differences in levels of Brd4, HEXIM1, Cyclin T1, or p-Cdk9 were observed in lysates of flavopiridol-treated cells versus control cells. This suggests that the dynamic shift of active P-TEFb from the large to the small complex may occur in nuclear speckles.
Fig. 7
Fig. 7
Active P-TEFb localizes to nuclear speckles in primary activated CD4+ T lymphocytes. A: Resting CD4+ T lymphocytes were cytospun onto glass coverslips and fixed immediately (upper parts) or activated (1 h) with PMA + ionomycin, cytospun onto glass coverslips and fixed (lower parts). The fixed cells were triple immunolabeled for SC35 (blue), Cyclin T1 (red), and p-Cdk9 (green), and the DNA was counterstained with DAPI to visualize nuclei. As can be seen, both the level of Cdk9 T-loop phosphorylation and the expression of Cyclin T1 and SC35 are highly up-regulated after 1 h of cell activation (compare upper to lower parts). B: Merge parts of the activated cells in A, showing the areas of co-localization between SC35 with Cyclin T1 (indicated by purple), SC35 with p-Cdk9 (indicated by aqua), Cyclin T1 with p-Cdk9 (indicated by yellow) and the co-localization of all three proteins (indicated by white). Ascan be seen, almost all of the active P-TEFb (CyclinT1/p-Cdk9) islocated either within or at the periphery of nuclear speckles in activated Tlymphocytes (merge part). This is particularly evident in the enlargement of a speckle region from one of these cells (boxed area on merge part) shown at the right of the merge part. The boxed region has been rotated 90° clockwise in the speckle enlargement for aesthetics. Bar, 5 µm.

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