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First published online 15 August 2006
doi: 10.1242/jcs.03104

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Phospho-specific binding of 14-3-3 proteins to phosphatidylinositol 4-kinase III ß protects from dephosphorylation and stabilizes lipid kinase activity

Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

View larger version (43K): [in a new window]   Fig. 1. PKD-mediated phosphorylation of PI4KIIIß at Ser294 promotes binding of 14-3-3 proteins. (A) Sequence alignment of the PKD consensus motif in PI4KIIIß from various species demonstrates conservation of a putative 14-3-3 binding motif. Phosphorylation of the serine is predicted to be required for recognition. (B) A Flag-tagged human wildtype PI4KIIIß expression vector was transiently transfected into HEK293 cells. Lysates were incubated with GST-14-3-3ß, , , , , , or GST coupled to Glutathione sepharose beads and bound proteins were separated by SDS-PAGE. Western blotting using a Flag-specific antibody detected the PI4KIIIß. PI4KIIIß expression was verified by immunoblotting of total cell lysates (TCL) with Flag-specific antibody. (C) Left panel: HEK293 cells transiently transfected with Flag-PI4KIIIß were treated for 1 and 2 hours with 100 nM OA solubilized in dimethyl sulfoxide. Total cell lysates (TCL) were immunoblotted and probed for phosphorylation of Ser294 with the PKD pMOTIF antibody, expression of Flag-PI4KIIIß was controlled with anti-Flag antibodies. The solvent dimethyl sulfoxide alone had no effect on the Ser294 phosphorylation. Right panel: Lysates of HEK293 cells transiently transfected with Flag-PI4KIIIß wildtype or the S294A mutant were subjected to Western blot and probed with the anti-PKD pMOTIF antibody. Expression of Flag-PI4KIIIß was controlled with anti-Flag antibodies. (D) Flag-tagged human wildtype and mutated PI4KIIIß expression vectors were transiently transfected into HEK293 cells. Cells were treated with 100 nM OA for 2 hours. Lysates were incubated with Glutathione beads coupled to GST-14-3-3 and bound proteins were separated by SDS-PAGE. Western blotting using a Flag-specific antibody detected PI4KIIIß. The expression level of PI4KIIIß was verified by immunoblotting of TCL. (E) Left panel: Flag-tagged wildtype and S294A PI4KIIIß expression vectors were transiently transfected into HEK293 cells along with HA-tagged 14-3-3, Glu-Glu-tagged 14-3-3 and 14-3-3, respectively. PI4KIIIß was precipitated using Flag-specific antibodies and protein complexes immunoblotted with Flag- and 14-3-3-specific antibodies. Total cell lysates were analysed in parallel to estimate expression levels. Right panel: Flag-tagged wildtype and S294A PI4KIIIß expression vectors were transiently transfected into HEK293 cells. 14-3-3 proteins were precipitated using 14-3-3-specific antibodies and protein complexes immunoblotted with Flag- and 14-3-3-specific antibodies. Total cell lysates were analysed in parallel to estimate expression levels. PD, pulldown.

View larger version (37K): [in a new window]   Fig. 2. DN-HA-14-3-3 proteins form inactive dimers and cannot interact with PI4KIIIß. (A) Left panel: HEK293 cells were cotransfected with plasmids encoding wildtype YN-14-3-3/YC-14-3-3 (red) or YN-14-3-3/YC-DN-14-3-3 (blue). Cells were harvested 48 hours after transfection in PBS supplemented with 5% FCS and 0.05% sodium azide, and BiFC was analysed by fluorescence flow cytometry. Untransfected cells were used as a negative control (filled). The data are presented in a histogram depicting EYFP fluorescence (FL1) (x axis) vs. cell number (count) (y axis). The results are representative of three independent experiments. Right panel: COS7 cells were cotransfected with the indicated plasmids. Cells were fixed 2 days after transfection and BiFC was analysed with confocal microscopy. A single optical section is shown. (B) Flag-tagged wildtype PI4KIIIß expression vectors were transiently transfected into HEK293 cells. Cells were lysed and Flag-PI4KIIIß proteins were precipitated with wildtype GST-14-3-3 or DN-GST-14-3-3 coupled Glutathione sepharose beads, resolved by SDS-PAGE and detected with Flag-specific antibodies. Equal amounts of GST-fusion proteins were visualized with anti-GST antibodies. (C) Left panel: Flag-tagged wildtype PI4KIIIß expression vector was transiently transfected into HEK293 cells along with HA-tagged 14-3-3 or HA-tagged DN-14-3-3, respectively. PI4KIIIß was precipitated using Flag-specific antibodies and protein complexes immunoblotted with a HA-specific monoclonal antibody. To verify HA-14-3-3 expression, total cell lysates were immunoblotted with Flag- and HA-specific antibodies. Right panel: HEK293 cells were transfected with HA-tagged wildtype or DN-14-3-3 expression vectors. 14-3-3 was precipitated using HA-specific antibodies and protein complexes immunoblotted with a PI4KIIIß-specific monoclonal and a 14-3-3-specific polyclonal antibody. Bar, 10 µm. PD, pulldown.

View larger version (56K): [in a new window]   Fig. 3. Analysis of the PI4KIIIß-14-3-3 interaction by BiFC with fluorescence flow cytometry and confocal microscopy. (A) HEK293 cells were cotransfected with plasmids encoding wildtype YN-PI4KIIIß/YC-14-3-3 (red) or YN-PI4KIIIß/YC-DN-14-3-3 (blue). Cells were harvested 48 hours after transfection in PBS supplemented with 5% FCS and 0.05% sodium azide and BiFC was analysed by fluorescence flow cytometry. Untransfected cells were used as a negative control (filled). The data are presented in a histogram depicting EYFP fluorescence (FL1) (x axis) vs cell number (count) (y axis). The results are representative of three independent experiments. (B) COS7 cells were cotransfected with YN-PI4KIIIß and YC-14-3-3. Cells were fixed two days after transfection, stained with anti-p230 followed by anti-mouse IgG Cy5. BiFC is shown in green, p230 proteins in red. Shown is a single optical section. Bar, 10 µm.

View larger version (68K): [in a new window]   Fig. 4. The binding of 14-3-3 proteins to serine 294 does not regulate nucleo-cytoplasmic shuttling of PI4KIIIß. (A) COS7 cells were transfected with wildtype Flag-PI4KIIIß or Flag-PI4KIIIßS294A. Cells were incubated with the vehicle (0.1% ethanol, control) or treated for 16 hours with 10 ng/ml leptomycin B. After incubation cells were fixed and stained with anti-Flag followed by anti-mouse IgG Alexa488. (B) COS7 cells were transfected with either Flag-PI4KIIIß alone (upper panel) or together with DN-HA-14-3-3 (lower panel). After 24 hours' incubation, cells were fixed and stained with anti-Flag followed by anti-mouse IgG Alexa488 and anti-14-3-3 followed by anti-rabbit IgG Alexa546. Flag-PI4KIIIß is stained in green, 14-3-3 proteins in red. Arrowheads indicate double-transfected cells. A series of images was taken at 0.5-µm intervals through the Z plane of the cell and were processed to form a projected image. Bar, 10 µm.

View larger version (38K): [in a new window]   Fig. 5. The binding of 14-3-3 proteins to PI4KIIIß stabilizes Ser294 phosphorylation. (A) Flag-tagged wildtype PI4KIIIß expression vector was transiently transfected into HEK293 cells along with vector, HA-tagged 14-3-3 or HA-tagged DN-14-3-3, respectively. Flag-PI4KIIIß was precipitated using Flag-specific antibodies and precipitates were immunoblotted with pMOTIF, Flag and HA-specific antibodies. To verify HA-14-3-3 expression total cell lysates (TCL) were immunoblotted with HA-specific antibodies. (B) HEK293 cells were transfected with plasmids encoding the indicated proteins, lysed and PKD1-GFP was precipitated using anti-GFP antibodies. Precipitates were subjected to in vitro kinase assay as described in Materials and Methods. (C) HEK293 cells were transfected with the Flag-PI4KIIIß expression construct and Flag-PI4KIIIß was purified with Flag-M2-Agarose. The purified enzyme was either incubated with PBS, 4 µg of GST, 4 µg of GST-DN-14-3-3 or 1 µg and 4 µg of GST-14-3-3 on ice overnight. The mixture was then incubated with or without 10 units of phosphatase, and dephosphorylation was performed at 30°C for 30 minutes. The phosphorylation status of Ser294 in Flag-PI4KIIIß was detected by immunoblotting with anti-PKD pMOTIF antibody; blot was further detected for Flag-PI4KIIIß. GST fusion proteins were visualized by Ponceau-S staining.

View larger version (49K): [in a new window]   Fig. 6. The binding of 14-3-3 proteins to PI4KIIIß maintains lipid kinase activity. HEK293 cells were transfected with Flag-PI4KIIIß together with wildtype or DN-HA-14-3-3 proteins. Treatment with OA was at 100 nM for 2 hours. Control samples were incubated with the vehicle (0.1% dimethyl sulfoxide). Flag-PI4KIIIß was precipitated using Flag-specific antibodies and lipid kinase assay was performed as described in Materials and Methods. Total cell lysates (TCL) were immunoblotted and probed for phosphorylation of Ser294 with the PKD pMOTIF antibody, equal amounts of protein were controlled with anti-Flag and anti-HA antibodies. Shown is one representative experiment (n=3). Density of spots representing PtdIns(4)P from the individual experiments was quantified using ImageQuant software (GE Healthcare). Respective vector controls were set as 100. The mean values were calculated for these three independent experiments and are shown in a graphic with associated errors (s.d.).