Phosphoinositide-dependent phosphorylation of PDK1 regulates nuclear translocation - PubMed (original) (raw)

Phosphoinositide-dependent phosphorylation of PDK1 regulates nuclear translocation

Michael P Scheid et al. Mol Cell Biol. 2005 Mar.

Abstract

3-phosphoinositide-dependent kinase 1 (PDK1) phosphorylates the activation loop of a number of protein serine/threonine kinases of the AGC kinase superfamily, including protein kinase B (PKB; also called Akt), serum and glucocorticoid-induced kinase, protein kinase C isoforms, and the p70 ribosomal S6 kinase. PDK1 contains a carboxyl-terminal pleckstrin homology domain, which targets phosphoinositide lipids at the plasma membrane and is central to the activation of PKB. However, PDK1 subcellular trafficking to other compartments is not well understood. We monitored the posttranslational modifications of PDK1 following insulin-like growth factor 1 stimulation. PDK1 underwent rapid and transient phosphorylation on S396, which was dependent upon plasma membrane localization. Phosphorylation of S396 was necessary for nuclear shuttling of PDK1, possibly through its influence on an adjacent nuclear export sequence. Thus, mitogen-stimulated phosphorylation of PDK1 provides a means for directed PDK1 subcellular trafficking, with potential implications for PDK1 signaling.

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Figures

FIG. 1.

IGF-1-stimulated nuclear shuttling of endogenous PDK1. (A) MCF-7 cells were plated onto glass coverslips and serum starved for 18 h. Cells were either stimulated with IGF-1 for 30 min or treated with leptomycin-B (50 nM) for 3 h. Cells were fixed in 3% paraformaldehyde, stained with anti-PDK1 (Cell Signaling), and counterstained with Alexa-488-conjugated anti-rabbit immunoglobulin G (IgG) (Molecular Probes). DNA was stained with DAPI (4′,6′-diamidino-2-phenylindole). Images were collected on a Zeiss LSM 510 confocal microscope. (B) MCF-7 cells growing in complete medium were fractionated into cytoplasmic (C) and nuclear (N) fractions as described in Materials and Methods. Portions of each fraction were resolved by SDS-PAGE and immunoblotted with anti-PDK1 (Cell Signaling).

FIG. 2.

IGF-1-stimulated PDK1 mobility shift. (A) HEK 293 cells were transfected with 500 ng of myc-PDK1 or empty vector for 24 h. Cells were starved for 18 h in serum-free medium and then stimulated with IGF-1 (100 ng/ml) or vehicle alone for 15 min. Cells were harvested into ice-cold lysis buffer and clarified by centrifugation, as described in Materials and Methods. PDK1 was immunoprecipitated with anti-myc 9E10 antibody, washed, and resuspended in CIP buffer (New England Biolabs). CIP (10 U) was added where indicated, and samples were incubated for 10 min at room temperature. Reactions were terminated by the addition of SDS sample buffer and boiling for 5 min. PDK1 was fractionated by SDS-PAGE and immunoblotted with 9E10 antibody. (B) HEK 293 cells were transfected with myc-PDK1 for 24 h, followed by serum starvation for 18 h, and then treated with IGF-1 (100 ng/ml) for the indicated times. Cells were lysed in ice-cold lysis buffer, and portions of the lysates were fractionated by SDS-PAGE, followed by immunoblotting with 9E10 anti-myc antibody. Samples were also probed with anti-phospho-T308 PKB antibody (Cell Siganling). (C) HEK 293 cells were serum starved for 18 h, followed by treatment with IGF-1 for the indicated times. Cells werelysed, and solubilized proteins were fractionated by SDS-PAGE. Endogenous PDK1 was detected with an anti-PDK1 antibody (Cell Signaling). Samples were also probed with an anti-phospho-S473 PKB antibody (Cell Signaling). (D) HEK 293 cells were transfected with 500 ng of myc-PDK1 for 24 h and then serum starved for 18 h. Cells were then treated where indicated with LY294002 for 15 min, followed by stimulation with IGF-1 (100 ng/ml) where indicated. myc-PDK1 was immunoprecipitated, fractionated by SDS-PAGE, and detected with anti-myc 9E10 antibody. (E) FLAG-PDK1 (3×; 200 ng) and p110-CAAX (200 ng) or empty vector was cotransfected into HEK 293 cells for 24 h. Cells were serum starved for 18 h and then stimulated with IGF-1 (100 ng/ml) for 15 min. Cells were lysed in ice-cold lysis buffer and fractionated by SDS-PAGE. Membranes were probed with anti-FLAG (M2 monoclonal; Sigma) or anti-p110 (Upstate). (F and G) 293 cells were cotransfected with myc-PDK1 (200 ng) and either RasN17, RasV12, or empty vector (200 ng each) where indicated for 24 h. Cells were serum starved for 18 h and then treated with LY294002 (25 μM) in the lanes indicated, followed by stimulation with IGF-1 (100 ng/ml) for 15 min where indicated. myc-PDK1 was immunoblotted as described above. (H) MCF-7 cells were serum starved overnight and treated with staurosporine (100 nM) for 10 min. Cells were stimulated with IGF-1 (100 ng/ml) for 15 min and then lysed in ice-cold lysis buffer. Clarified lysates were fractionated by SDS-PAGE, and endogenous PDK1 was detected with anti-PDK1 antibody (Cell Signaling). Phospho-T308, S473, and total PKB (also from Cell Signaling) were also detected.

FIG. 3.

PI3K and PH domain requirement for PDK1 phosphorylation. (A and B) HEK 293 cells were transfected with 500 ng of myc-PDK1 for 24 h and then serum starved for 18 h. Cells were labeled with [32P]orthophosphate (1 mCi/ml) in phosphate-free medium for 4 h, followed by treatment with LY294002 (25 mM) or rapamycin (100 nM) for 30 min. Following treatment with IGF-1 for 15 min and solubilization in ice-cold lysis buffer, PDK1 was immunoprecipitated with anti-myc 9E10 antibody and fractionated by SDS-PAGE. (C) Cells were transfected with wild-type PDK1 or R474A-PDK1, labeled, and stimulated with IGF-1 as described above (top). A portion of the immunoprecipitate was used to probe total PDK1 (bottom). (D) MCF-7 cells were plated onto glass coverslips and transfected with myc-PDK1 or myc-R474A-PDK1 for 24 h. Cells were then serum starved for 18 h and treated with IGF-1 for 15 min. Cells were fixed in 3% paraformaldehyde, stained with anti-myc 9E10 antibody, and counterstained with Alexa-488-conjugated anti-mouse antibody (Molecular Probes). Samples were visualized by confocal microscopy.

FIG. 4.

Myristoylation of PDK1 increases phosphorylation. (A) Myc-PDK1 or myr-R474A-PDK1 was transfected into HEK 293 cells for 24 h, followed by 18 h of serum starvation and treatment with IGF-1 for 15 min. Lysates were fractionated by SDS-PAGE, and PDK1 was detected with anti-PDK1 antibody (Cell Signaling Technologies). (B) HEK 293 cells transfected with myc-PDK1 or myr-R474A-PDK1 were labeled as described in the legend to Fig. 3 and stimulated with IGF-1 for 15 min. PDK1 was detected by autoradiography (top) and a portion of the immunoprecipitate was probed for total PDK1 (bottom). (C) myc-PDK1 or myr-R474A-PDK1 were transfected into MCF-7 cells growing on glass coverslips. After 24 h, cells were fixed in 3% paraformaldehyde, and PDK1 was stained with anti-PDK1 antibody (Cell Signaling Technologies) and counterstained with Alexa-488-conjugated anti-rabbit IgG (Molecular Probes). Confocal images were captured with a Zeiss LSM 510 microscope. (D) HEK 293cells transfected with myc-PDK1 or myr-R474A-PDK1 were harvested and fractionated into cytoplasmic (C) or membrane (M) fractions as described in Materials and Methods. Volumes of the lysates corresponding to equal cell equivalents were fractionated by SDS-PAGE and immunoblotted with anti-PDK1 antibody (Cell Signaling Technologies). (E) myc-PDK1, myc-R474A-PDK1, or myr-R474A-PDK1 was expressed in HEK 293 cells for 24 h, followed by serum starvation for 18 h. Cells were labeled for 4 h with [32P]orthophosphate (1 mCi/ml), stimulated with IGF-1 (100 ng/ml) where indicated, and solubilized in ice-cold lysis buffer. PDK1 was immunoprecipitated with anti-myc 9E10 antibody and resolved by SDS-PAGE. 32P-labeled PDK1 was excised from the gel and digested with trypsin (Calbiochem) overnight at 37°C as described in Materials and Methods. The dried tryptic peptides were spotted onto cellulose plates and separated in the first dimension by electrophoresis in buffer (pH 1.9), dried, and separated in the second dimension with phosphochromatography buffer. 32P-labeled peptides were visualized by PhosphorImager analysis (Molecular Dynamics). (F) The tyrptic peptide labeled A was scraped from the cellulose plate and digested with 6 N constantly boiling HCl at 100°C for 20 min. The hydrolyzed amino acids were washed several times, separated by electrophoresis chromatography on cellulose plates in buffer (pH 1.9), and visualized by autoradiography. The migration of phosphoserine, phosphothreonine, and phosphotyrosine was determined by comigration with cold phosphoamino acids and visualized by ninhydrin staining.

FIG. 5.

PDK1 phosphorylation does not require S241 or kinase activity. (A) myc-PDK1 or myc-S241A-PDK1 was expressed in HEK 293 cells for 24 h, serum starved for 18 h, and stimulated where indicated with IGF-1 for 15 min. PDK1 was immunoprecipitated with anti-myc 9E10 antibody, fractionated by SDS-PAGE, and immunoblotted with anti-S241 antibody (Cell Signaling) (top) and anti-PDK1 (Cell Signaling) (bottom). (B) HEK 293 cells were transfected with myc-PDK1, myc-R474A-PDK1, myc-S241-PDK1, or empty vector for 24 h; serum starved for 18 h; and then stimulated with IGF-1 for 15 min. PDK1 was immunoprecipitated with anti-myc 9E10 antibody and assayed for activity in vitro with S422D SGK as substrate as described in Materials and Methods. Reactions were terminated by addition of 2× SDS sample buffer and boiling. Proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride, and visualized by autoradiography. (C) myc-PDK1 and myc-K111A-PDK1 were transfected into HEK 293 cells and labeled with [32P]orthophosphate as described in the legend to Fig. 3. (D) Kinase activity of myc-PDK1 and myc-K111A-PDK1 was measured as in the data shown in panel B. (E) Tryptic peptide mapping was performed on 32P-labeled PDK1 isolated from panel C.

FIG. 6.

PDK1 phosphorylation at S396 following IGF-1 stimulation. (A) myc-PDK1 or myc-S396A-PDK1 was transfected into 293 cells for 24 h, serum starved for 18 h, and stimulated with IGF-1 or pervanadate (PV) for 15 min. PDK1 was immunoprecipitated with anti-myc 9E10 antibody and resolved by SDS-PAGE. Immunoblotting was performed with anti-PDK1 (top) or anti-phosphotyrosine 4G10 (bottom) antibody. (B) HEK 293 cells were transfected with myc-PDK1 or myc-S396A-PDK1 and labeled with 3[32P]orthophosphate for 4 h. Cells were stimulated with IGF-1 for 15 min and solubilized in ice-cold lysis buffer. PDK1 was immunoprecipitated with anti-myc 9E10 antibody, digested with trypsin, and resolved by 2D chromatography as described in the legend to Fig. 4 and Materials and Methods. (C) HEK 293 cells transiently expressing myc-PDK1, myc-S396A-PDK1, or myc-S396D-PDK1 were stimulated with IGF-1 for 15 min. Lysates were fractionated by SDS-PAGE, and PDK1 was detected by immunoblotting with anti-PDK1 antibody (Cell Signaling). (D) HEK 293 cells were transfected with myc-PDK1 or myc-S396A-PDK1, followed by labeling with [32P]orthophosphate for 4 h and stimulation with IGF-1. myc-PDK1 was immunoprecipitated with anti-myc 9E10 antibody and fractionated by SDS-PAGE. 32P-labeled PDK1 was visualized and quantitated with a PhosphorImager (Molecular Dynamics). Error bars represent the standard deviation of three independent experiments.

FIG. 7.

2D tryptic mapping of in vitro-phosphorylated PDK1. (A) myc-PDK1 was expressed and immunoprecipitated from serum-starved HEK 293 cells. [γ-32P]ATP and MnCl2 were added, and samples were incubated at 30°C for 30 min. Reactions were stopped by the addition of 2× SDS sample buffer and boiling. PDK1 was fractionated by SDS-PAGE, excised from the gel, and digested with trypsin as described in Materials and Methods. Peptides were separated by chromatography and visualized with a PhosphorImager. The image on the left shows an in vivo-labeled PDK1 digestion. Spots labeled a, b, and c indicate areas of phosphorylation. (B) Comparison of rat, mouse, and human PDK1 sequence alignment surrounding the polyserine motif and S396. S393 has also been reported to be a phosphorylation site and is indicated. Potential phosphorylation sites carboxyl terminal to S396 are not conserved between species.

FIG. 8.

Nuclear PDK1 colocalizes with PKB and inhibits FOXO3a transcriptional activity and nuclear localization. (A) Wild-type PDK1 or mNES-PDK1 (50 ng) was cotransfected with HA-PKB (50 ng) on glass coverslips for 24 h. PDK1 and PKB were stained with anti-PDK1 (Cell Signaling Technologies) and anti-HA antibodies and visualized by confocal microscopy. (B) Cells were transfected with forkhead transcription factor FKHRL1 (FOXO3a; 25 ng), pGL2-Luciferase FOXO reporter plasmid (100 ng), and the indicated concentrations of wild-type PDK1. β-Galactosidase was coexpressed as a control for transfection. After 24 h of luciferase activity was measured and normalized to β-galactosidase activity. (C) Wild-type PDK1 or mNES-PDK1 at the indicated amounts (in nanograms) was cotransfected with FKHRL1 (FOXO3a; 25 ng) and the FOXO-responsive luciferase reporter plasmid (100 ng). After 24 h, luciferase activity was measured and normalized to β-galactosidase activity (top). Portions of the reserved lysates were immunoblotted for PDK1 (bottom). (D) FOXO3a and either empty vector, wild-type PDK1, or mNES-PDK1 where indicated were cotransfected into MCF-7 cells plated on glass coverslips. After 24 h, cells were treated for 2 h with LY294002 (25 μM) where indicated. Cells were stained with anti-FKHR and anti-PDK1 (both from Cell Signaling Technologies) and visualized by confocal microscopy.

FIG. 8.

FIG. 9.

Effect of leptomycin-B and NES mutation of PDK1 on nuclear shuttling. (A) MCF-7 cells were plated on glass coverslips and transfected with myc-PDK1 or myc-S396A-PDK1 for 24 h. Cells were then treated with leptomycin-B for 3 h where indicated. Cells were fixed in 3% paraformaldehyde and stained with anti-myc 9E10 antibody, followed by Alexa-488-conjugated anti-mouse IgG (Molecular Probes). Images were collected with a Zeiss LSM 510 confocal microscope. (B) mNES-PDK1 or mNES-PDK1 containing various secondary mutations was transfected into MCF-7 cells growing on glass coverslips for 24 h. Cells were fixed with 3% paraformaldehyde, stained with anti-myc 9E10, and visualized as described above.

FIG. 10.

Nuclear shuttling of PDK1 requires S396. (A) PTEN−/− MEFs were plated on glass coverslips and transfected with myc-PDK1 or myc-S396A-PDK1 (100 ng) for 24 h, followed by serum starvation for 18 h. Cells were stimulated with PDGF (50 ng/ml) for the indicated times and fixed with 3% paraformaldehyde. Cells were stained with anti-PDK1 (Cell Signaling Technologies) and Alexa-488 conjugated to anti-rabbit IgG and examined by laser scanning confocal microscopy. (B) MCF-7 cells growing on glass coverslips were transfected with myc-PDK1 or myc-S396A-PDK1 (each, 100 ng) and stimulated with IGF-1 for 30 min. PDK1 was visualized as described for panel A. (C) Enhanced GFP-PDK1 or GFP-S396A-PDK1 was transfected into MCF-7 cells plated on glass coverslips. Transfected cells were serum starved for 18 h and then treated with IGF-1 (100 ng/ml) for 30 min. Cells were fixed with 3% paraformaldehyde and visualized by laser-scanning confocal microscopy. (D) Quantitation of nuclear PDK1 staining from a sample of MCF-7 cells treated as in panel B. Total nuclear fluorescence was determined with Zeiss LSM software, and the average value for the indicated number of cells examined is shown. The asterisk indicates significant difference with a P value of <0.0001 as determined with Student's paired t test.

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Phosphoinositide-dependent phosphorylation of PDK1 regulates nuclear translocation - PubMed (original) (raw)