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First published online 15 August 2006
doi: 10.1242/jcs.03090

Journal of Cell Science 119, 3676-3685 (2006)
Published by The Company of Biologists 2006
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Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23

Sandeep S. Negi and Mark O. J. Olson*

Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS 39216, USA


Figure 1
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  		Fig. 1. Structural features and potential phosphorylation sites in protein B23.1. (A) The CK2 site lies in the first acidic region. The potential CDK1 sites (arrowheads) are located in or near the nucleic acid binding region. Other functional segments are indicated below the diagram. (B) CK2 (Ser125) and potential CDK1 (Thr199, 219, 234 and 237) sites of phosphorylation on protein B23.1 are shown in bold.

 

Figure 2
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  		Fig. 2. Location of protein B23-GFP and mutants during interphase and mitosis. (A) Localization of GFP-tagged wild-type and endogenous protein B23 during different phases of mitosis. Both the proteins showed similar localization during various phases of mitosis. Upper panels, GFP-tagged expressed protein; lower panels, endogenous protein as detected by immunofluorescence using a monoclonal antibody. Arrows indicate the position of NDF in an anaphase cell and arrowheads indicate the position of PNBs in the newly forming nucleus at telophase. (B) Expression of the wild type protein and the mutants tagged with GFP. Immunoblot was developed with anti-GFP mAb. B23-GFP (lane 1), S125A-GFP (lane 2), S125E-GFP (lane 3) and untransfected cell lysate (lane 4). (C) Location of (a) wild-type B23-GFP, (b) S125A-GFP, (c) S125E-GFP and (d) endogenous B23 detected by immunofluorescence using a monoclonal antibody in interphase HeLa cells. All the fusion proteins localized to the nucleolus as did the endogenous B23 protein. Corresponding images of Hoechst 33342-stained DNA are shown in (a'), (b'), (c') and (d'). Bars, 10 µm.

 

Figure 3
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  		Fig. 3. Fluorescence recovery after photo bleaching (FRAP) analysis of GFP-tagged B23 and its CK2 phosphorylation mutants. Cells were transfected with GFP-expressing construct and subjected to photobleaching 24-36 hours post transfection. Five scans were taken before bleaching. An area in the nucleolus or a whole nucleolus was bleached with the 488 nm laser and the fluorescence recovery of the bleached spot was measured every 5 seconds. The normalized fluorescence recovery in the ROI at each time point after photobleaching was plotted against time. The t1/2s of recovery for the mutants and the wild type protein B23 are indicated in the figure. Error indicates s.d. of the mean for each fitted curve. At least eight datasets were analysed for each result.

 

Figure 4
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  		Fig. 4. The C-terminal nucleic-acid-binding region affects the mobility of the nucleoplasmin family proteins. (A) Diagram of nucleoplasmin family proteins. B23.2 and NPM3 lack the C-terminal nucleic-acid-binding region of B23.1. The position of the CK2 and CDK phosphorylation sites (Thr199, 219, 234 and 237) are indicated by an arrowhead and arrows, respectively. (B) Representative image showing distribution of GFP-B23.1, GFP-B23.2 and GFP-NPM3 in the nuclei of HeLa cells. The cells were transiently transfected with the corresponding plasmid and observed after 36 hours under a Leica confocal microscope. Relative amounts of B23.1, B23.2 and NPM3 present in the nucleolus and nucleoplasm are shown in the box. (C) Fluorescence recovery after photobleaching (FRAP) analysis of GFP-tagged nucleoplasmin family proteins. Cells were transfected with GFP-expressing construct and subjected to photobleaching 24-36 hours post transfection using a 488 nm laser. Five scans were taken before bleaching. An area in the nucleolus or a whole nucleolus was bleached and the fluorescence recovery of the bleached spot was measured every 5 seconds. Normalized fluorescence recovery in the ROI at each time point after photobleaching was plotted against time. The t1/2s of recovery for the nucleoplasmin family proteins are indicated in the figure. Error indicates s.d. of the mean for each fitted curve.

 

Figure 5
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  		Fig. 5. Timing of phosphorylation of CDK-type sites in protein B23.1 during the cell cycle. HeLa cells were released from double thymidine block and harvested at indicated time points for (A) FACS and (B) western blot analysis. (A) Cells were fixed in methanol and treated with 5 µg/ml RNase A followed by propidium iodide staining for FACS analysis. Maximum percentage of cells in G2-M phase was reached 8 hours after release from double thymidine block. (B) For western blot, an equal amount of protein was loaded in each lane and then probed with either polyclonal antibody against T199-P (T199p) and T234/237-P (T234/237p) or monoclonal antibody against protein B23. (C) HeLa cells were blocked in mitosis by nocodazole treatment followed by incubation in DMSO or 75 µM roscovitine for 30 minutes. For phosphatase inhibitor treatment, nocodazole-treated cells were incubated with different concentrations of okadaic acid or calyculin A for 30 minutes followed by 75 µM roscovitine treatment. The western blot was probed with Thr199-P (T199p), Thr234/237-P (T234/237p) and B23 antibodies. Lanes: (1) DMSO, (2) Roscovitine (75 µM) treatment, (3) okadaic acid (OA, 500 nM) followed by roscovitine treatment, (4) okadaic acid (OA, 5 nM) followed by roscovitine treatment, (5) calyculin A (Cal A, 5 nM) followed by roscovitine treatment.

 

Figure 6
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  		Fig. 6. Location of protein B23 containing Thr199-P during mitosis. Indirect immunofluorescence studies were conducted using the B23 Thr199-P antibody (T199p) to study its location during different phases of mitosis. Cells stained with the DNA-binding dye Hoechst 33342, in different stages of mitosis are indicated by arrows. (A) B23 was phosphorylated at Thr199 at the onset of mitosis as seen by the signal for Thr199-P antibody in early mitotic cells. The surrounding interphase cells showed weak or no signal with this antibody. (B) The signal for Thr199-P antibody was detected during early anaphase but it disappeared as the cell progressed through mid anaphase. In the same panel, one of the cells in metaphase was stained by Thr199-P antibody but no signal was detected in the surrounding interphase cells. (C) With B23 antibody staining, arrowheads and arrows show NDF and PNBs in anaphase and telophase respectively. Corresponding images show that the Thr199-P staining is absent in these cells. Bars, 10 µm.

 

Figure 7
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  		Fig. 7. Location and mobility of the CDK1 phosphorylation-mimicking mutant. (A) Location of T4E-GFP mutant in interphase HeLa cells. T4E-GFP transfected cells were analyzed after 36 hours under a fluorescence microscope. This mutant shows a similar distribution in the nucleus to the nucleoplasmin family proteins, B23.2 and NPM3 lacking the C-terminus. Bar, 10 µm. (B) Expression of T4E-GFP mutant in interphase HeLa and CMT3 cells. The western blot was developed with anti-GFP monoclonal antibody. (Lane 1) untransfected cell lysate; (lane 2) T4E-GFP transfected HeLa cell lysate; (lane 3) T4E-GFP transfected CMT3 cell lysate. (C) Fluorescence recovery after photo bleaching (FRAP) analysis of GFP-tagged Thr199/219/234/237 Glu mutant B23. Cells were transfected with GFP-expressing construct and subjected to photobleaching 24-36 hours post transfection using a 488 nm laser. Five scans were taken before bleaching. An area in the nucleolus or a whole nucleolus was bleached and the fluorescence recovery of the bleached spot was measured every 5 seconds. Normalized fluorescence recovery in the ROI at each time point after photobleaching was plotted against time. The t1/2s of recovery for the T4E-GFP mutant and wild-type protein B23-GFP are indicated in the figure. Error indicates s.d. of the mean for each fitted curve.

 

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