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First published online January 30, 2004
doi: 10.1242/10.1242/jcs.00905

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Nuclear translocation of the Hsp70/Hsp90 organizing protein mSTI1 is regulated by cell cycle kinases

¹ Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa
² Division of Pathology, Institute of Ophthalmology, University College London, London, EC1V 9EL, UK
³ Division of Cell Biology, Institute of Ophthalmology, University College London, London, EC1V 9EL, UK

View larger version (69K): [in a new window]   Fig. 1. The subcellular localization of mSTI1-EGFP and NLSmSTI1-EGFP. (A) NIH3T3 fibroblasts were transiently transfected with pB-mSTI1-EGFP, fixed in 3.7% paraformaldehyde, permeabilized in 0.1% saponin (all panels) and immunostained with SF1 polyclonal rabbit anti-mSTI1 and Cy3-conjugated donkey anti-rabbit antibodies (middle and right panel). Cells were mounted and visualized by confocal laser fluorescence microscopy. Scale bars, 10 µm. (B) NIH3T3 fibroblasts were stably transfected with pB-EGFP (top), pB-mSTI1-EGFP (middle) and pB-NLSmSTI1-EGFP (bottom). Cells were fixed in 3.7% paraformaldehyde and stained with DAPI before visualization by confocal laser fluorescence microscopy. Scale bars, 10 µm.

View larger version (51K): [in a new window]   Fig. 2. The nuclear localization of mSTI1-EGFP is enhanced by inhibition of nuclear export and inhibition of cdc2 kinase but not by inhibition of CKII. (A) Untransfected NIH3T3 fibroblasts (left), NIH3T3 fibroblasts stably transfected with pBEGFP (middle) or with pB-mSTI1-EGFP (right) were exposed to leptomycin B (18.5 nM), olomoucine (0.1 mM) or DRB (0.3 mM) for 3 hours, 8 hours and 8 hours, respectively. Untransfected cells were immunostained to detect endogenous mSTI1 with SF1 polyclonal rabbit anti-mSTI1 and Cy3-conjugated donkey anti-rabbit antibodies, and transfected cells were fixed in 3.7% paraformaldehyde before mounting and visualization by confocal laser fluorescence microscopy. Scale bars, 10 µm. (B) Cells as above were exposed to leptomycin B, olomoucine and DRB, and cells demonstrating cytoplasmic fluorescence or nuclear and cytoplasmic fluorescence were quantified. The error bars represent standard deviations.

View larger version (55K): [in a new window]   Fig. 3. The nuclear localization of endogenous mSTI1 and mSTI1-EGFP is enhanced by G1/S-phase arrest. (A) Untransfected NIH3T3 fibroblasts (left), NIH3T3 fibroblasts stably transfected with pB-EGFP (middle) or with pB-mSTI1-EGFP (right) were exposed to hydroxyurea (10 mM) for 10 hours. Untransfected cells were immunostained to detect endogenous mSTI1 with SF1 polyclonal rabbit anti-mSTI1 and Cy3-conjugated donkey anti-rabbit antibodies, and transfected cells were fixed in 3.7% paraformaldehyde before mounting and visualization by confocal laser fluorescence microscopy. Scale bars, 10 µm. (B) Cells were exposed to hydroxyurea as above, and cells demonstrating cytoplasmic fluorescence or nuclear and cytoplasmic fluorescence were quantified. The bars represent standard deviations.

View larger version (32K): [in a new window]   Fig. 4. Phosphorylation mimic at S189 and not T198 affects the localization of mSTI1-EGFP. (A) NIH3T3 fibroblasts were stably transfected with pB-mSTI1-EGFP or derivative plasmids pB-mSTI1(S189A)-EGFP, pB-mSTI1(T198A)-EGFP, pB-mSTI1(S189E)-EGFP and pB-mSTI1(T198E)-EGFP. Cells were fixed in 3.7% paraformaldehyde before mounting and visualization by confocal laser fluorescence microscopy. Scale bars, 10 µm. (B) Cells were transfected as above, and cells demonstrating cytoplasmic fluorescence or nuclear and cytoplasmic fluorescence were quantified. The error bars represent standard deviations.

View larger version (51K): [in a new window]   Fig. 5. Phosphorylation mimic and phosphorylation site removal at T198 affects the localization of mSTI1-EGFP under G1/S-phase arrest. (A) NIH3T3 fibroblasts were stably transfected with pB-mSTI1-EGFP, derivative plasmids pB-mSTI1(T198E)-EGFP or with pB-mSTI1(T198A)-EGFP. Cells were treated with hydroxyurea (10 mM) for 10 hours and fixed in 3.7% paraformaldehyde before mounting and visualization by confocal laser fluorescence microscopy. Scale bars, 10 µm. (B) Cells were transfected as above, and cells demonstrating cytoplasmic fluorescence or nuclear and cytoplasmic fluorescence were quantified. The error bars represent standard deviations.

View larger version (41K): [in a new window]   Fig. 6. mSTI1-EGFP and its modified derivatives can interact with mSTI1, Hsp70 and Hsp90. Cells were transfected with pB-mSTI1-EGFP or derivative plasmids pB-mSTI1(S189A)-EGFP, pB-mSTI1(T198A)-EGFP, pB-mSTI1(S189E)-EGFP, pB-mSTI1(T198E)-EGFP or pB-EGFP. EGFP chimeric proteins were immunoprecipitated from transfected and control untransfected cell lysates with antibodies against GFP (anti-GFP) or non-immune IgG (IgG) and subjected to western blotting, as indicated. The mSTI1-EGFP and mSTI1 proteins were detected with a polyclonal antibody raised against GST-mSTI1 (Lässle et al., 1997). Hsp90 was detected with monoclonal antibody sc31119 and Hsc70 and Hsp70 were detected with monoclonal antibody BRM-22. The mSTI1, Hsp90 and Hsp70 proteins all specifically immunoprecipitated with the mSTI1-EGFP chimeric proteins.

View larger version (16K): [in a new window]   Fig. 7. mSTI1 is phosphorylated in vitro at multiple sites by cdc2 kinase. In vitro cdc2 kinase assays were incubated for 3 hours and 9 hours using purified recombinant mSTI1 and its modified derivatives (5 µM). The phosphorylated proteins were resolved by SDS-PAGE and the radioactive phosphorylation in the dried gel was detected by autoradiography.