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. 2000 Aug;20(15):5712-21.
doi: 10.1128/MCB.20.15.5712-5721.2000.

Effect of phosphoinositide-dependent kinase 1 on protein kinase B translocation and its subsequent activation

N Filippa  1 , C L Sable, B A Hemmings, E Van Obberghen
Affiliations

Effect of phosphoinositide-dependent kinase 1 on protein kinase B translocation and its subsequent activation

N Filippa et al. Mol Cell Biol. 2000 Aug.

Abstract

In this report we investigated the function of phosphoinositide-dependent protein kinase 1 (PDK1) in protein kinase B (PKB) activation and translocation to the cell surface. Wild-type and PDK1 mutants were transfected into HeLa cells, and their subcellular localization was analyzed. PDK1 was found to translocate to the plasma membrane in response to insulin, and this process did not require a functional catalytic activity, since a catalytically inactive kinase mutant (Kd) of PDK1 was capable of translocating. The PDK1 presence at the cell surface was shown to be linked to phospholipids and therefore to serum-dependent phosphatidylinositol 3-kinase activity. Using confocal microscopy in HeLa cells we found that PDK1 colocalizes with PKB at the plasma membrane. Further, after cotransfection of PKB and a PDK1 mutant (Mut) unable to translocate to the plasma membrane, PKB was prevented from moving to the cell periphery after insulin stimulation. In response to insulin, a PKB mutant with its PH domain deleted (DeltaPH-PKB) retained the ability to translocate to the plasma membrane when coexpressed with PDK1. Finally, we found that DeltaPH-PKB was highly active independent of insulin stimulation when cotransfected with PDK1 mutants defective in their PH domain. These findings suggest that PDK1 brings PKB to the plasma membrane upon exposure of cells to insulin and that the PH domain of PDK1 acts as a negative regulator of its enzyme activity.

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Figures

FIG. 1
FIG. 1
Effect of insulin on localization of PDK1 mutants. HeLa cells were transfected with 4 μg of either wild-type (Wild), myristoylated (Myr), or kinase dead (Kd) PDK1 as described in Materials and Methods. Forty-eight hours later, HeLa cells were incubated for 5 min with buffer or insulin (10−6 M). Cells were washed and fixed with paraformaldehyde (4%) prior to incubation with an antibody to Myc followed by incubation with a Texas red-linked mouse antibody to label PDK1 mutants. Slides were mounted and analyzed by confocal microscopy. Images represent the center section of the x-y plane.
FIG. 2
FIG. 2
Effect of kinase-dead PDK1 on PKB activation. (A) Myc-PDK1 constructs (Wild and Kd) were expressed separately in 293 cells. Immunoprecipitates and PDK1 purified from baculovirus were incubated with [γ-32P]ATP (20 min at 30°C) and ΔPH-PKB purified from baculovirus-expressing Sf9 cells. Samples were subjected to SDS–10% PAGE under reducing conditions followed by autoradiography. Arrows point to the positions of Myc-PDK1, PDK1, and ΔPH-PKB. Asterisks indicate proteins purified from baculovirus-expressing Sf9 cells. (B) 293 cells were transfected with wild-type PKB and, where indicated, with PDK1 constructs. Serum-starved cells were incubated for 5 min in the absence (white bars) or presence (black bars) of insulin (10−6 M). After incubation, cells were lysed, PKB was immunoprecipitated, and its kinase activity was determined using Crosstide as described in Materials and Methods. PKB activity is expressed as fold stimulation compared to basal levels, and the corresponding value is indicated above each column. Values shown are representative of at least four independent experiments performed in triplicate. The levels of the expressed enzymes measured by immunoblotting are shown below the bar graph. (C) HeLa cells were transfected with wild-type PKB and, where indicated, with PDK1 constructs. Serum-starved cells were incubated for 5 min with buffer (B) or insulin (I). After incubation, cells were lysed and 50 μg of the lysates was subjected to SDS-PAGE. PKB phosphorylation was determined by immunoblot analysis with an antibody specifically recognizing phospho-Thr308.
FIG. 3
FIG. 3
Role of phospholipids in PDK1 localization and PKB basal activation. (A) Wild-type PDK1 (Wild) was expressed in HeLa cells and after 24 h, cells were serum starved with either 0.2% (wt/vol) BSA for 12 h or 0.1% (wt/vol) BSA in DMEM for 24 h. Cells were washed and fixed with paraformaldehyde (4%) prior to incubation with an antibody to Myc followed by incubation with a Texas red-linked mouse antibody to label PDK1. Slides were mounted and analyzed by confocal microscopy. Images represent the center section of the x-y plane. (B) 293 cells were transfected with wild-type PKB and PDK1 mutants. Cells were serum starved with 0.1% (wt/vol) BSA and incubated for 5 min in buffer (white bars) or insulin (10−6 M) (black bars). Then, PKB activity was determined as described in Materials and Methods. PKB activity is expressed as fold stimulation compared to basal levels, and the corresponding value is indicated above each column. Values shown are representative of three independent experiments performed in triplicate. The levels of the expressed enzymes measured by immunoblotting are shown below the bar graph. (C) 293 cells were serum starved in either 0.2% (wt/vol) or 0.1% (wt/vol) BSA for 24 h prior to exposure for 5 min to insulin (I) (10−6 M) or to buffer. Total cellular protein lysates were subjected to immunoprecipitation with an antibody to IRS-1, and associated PI 3-kinase activity was measured with PtdIns or PtdIns-4-P plus PtdIns-4,5-P2 as substrates. The position of 32P-labeled Pi (PIP) is indicated. Radioactivity associated with each spot was quantified using phosphorimaging and is indicated below the autoradiograph. (D) HeLa cells were transfected with wild-type PKB and where indicated with PDK1 constructs. Cells were serum starved with either 0.2% (wt/vol) BSA for 12 h or 0.1% (wt/vol) BSA for 24 h and incubated for 5 min with buffer or insulin (I). After incubation, cells were lysed and 50 μg of the lysates was subjected to SDS-PAGE. PKB phosphorylation was revealed by immunoblotting with an antibody specific for phospho-Thr308 peptide. (E) 293 cells were transfected with Myr-PKB or wild-type PKB and, where indicated, with Δp85. Cells were serum starved and lysed, PKB was immunoprecipitated, and its activity was determined as described in Materials and Methods.
FIG. 4
FIG. 4
Effect of PDK1 mutants on PKB subcellular localization and activation. (A) GFP-PKB was coexpressed in HeLa cells together with wild-type PDK1 (Wild), myristoylated PDK1 (Myr), or PDK1 point mutated in its PH domain (Mut). Forty-eight hours later, HeLa cells were exposed for 5 min to buffer or to insulin (10−6 M), washed, and fixed with paraformaldehyde (4%) prior to incubation with an antibody to Myc, followed by an incubation with a Texas red-linked mouse antibody to label PDK1 constructs. Slides were mounted and analyzed by confocal microscopy. Images represent the center section of the x-y plane. (B) HeLa cells were transfected with wild-type PKB and wild-type PDK1 (Wild) or Myr-PDK1 (Myr). Cells were serum starved in 0.2% (wt/vol) BSA and incubated for 5 min in the presence (black bars) or absence (white bars) of insulin (10−6 M). Then, PKB activity was determined as described in Materials and Methods. PKB activity is expressed as fold stimulation compared to buffer conditions, and the corresponding value is indicated above each column. Values shown are representative of at least three independent experiments performed in triplicate. The levels of the expressed enzymes measured by immunoblotting are shown below the bar graph. (C) 293 cells were transfected with HA-PKB or both Myc-PDK1 and HA-PKB. The cytosolic (S100) and particulate (P100) fractions were prepared as described in Materials and Methods. The PKB phosphorylation state was revealed by immunoblotting with an antibody specific for phospho-Thr308 peptide. (D) COS-7 cells were serum starved in 0.2% (wt/vol) BSA and incubated for 5 min with insulin (I) (10−6 M) or buffer (B). Then, cytosolic (S100) and particulate (P100) fractions were prepared as described in Materials and Methods. PKB was immunoprecipitated (IP) from 500 μg of the cytosolic or particulate fraction, and changes in PKB and PDK1 distributions were detected by immunoblotting (IB) with antibodies to PKB or PDK1. PKB phosphorylation was revealed by immunoblotting with an antibody specific for phospho-Thr308 peptide.
FIG. 5
FIG. 5
Effect of PDK1 mutants on ΔPH-PKB subcellular localization and activation. (A) GFP–ΔPH-PKB was coexpressed in HeLa cells together with wild-type PDK1 (Wild), myristoylated PDK1 (Myr), or PDK1 point mutated in its PH domain (Mut). Forty-eight hours later, HeLa cells were exposed for 5 min to buffer or to insulin (10−6 M), washed, and fixed with paraformaldehyde (4%) prior to incubation with an antibody to Myc, which was followed by an incubation with a Texas red-linked mouse antibody to label PDK1 constructs. Slides were mounted and analyzed by confocal microscopy. Images represent the center section of the x-y plane. (B) 293 cells were transfected with ΔPH-PKB and wild-type PDK1 (Wild) or Myr-PDK1 (Myr). Cells were serum starved in 0.2% (wt/vol) BSA and incubated for 5 min in the presence (black bars) or absence (white bars) of insulin (10−6 M). Then, PKB activity was determined as described in Materials and Methods. PKB activity is expressed as fold stimulation compared to buffer conditions, and the corresponding value is indicated above each column. Values shown are representative of at least three independent experiments performed in triplicate. HeLa cells were transfected with 8 μg of ΔPH-PKB construct as described in Materials and Methods. Forty-eight hours later, cells were stimulated for 5 min with insulin (10−6 M). Cells were washed and fixed with paraformaldehyde (4%) prior to incubation with WGA-rhodamine (red) to label the plasma membrane. Slides were mounted and analyzed by confocal microscopy. Images represent the center section of the x-y plane. Membrane staining is in red, and GFP is in green. Areas of colocalization appear as yellow. The levels of the expressed enzymes measured by immunoblotting are shown below the bar graph. (C) 293 cells were transfected with ΔPH-PKB with or without Mut-PDK1. Anti-HA (ΔPH-PKB) immunoprecipitates were resolved by SDS-PAGE followed by Myc (Mut-PDK1) immunoblotting. Whole-cell lysates (100 μg) were resolved; Mut-PDK1 and ΔPH-PKB expression are detected using the Myc and HA antibodies, respectively.
FIG. 6
FIG. 6
Role of the PDK1 PH domain in PKB activation. (A) PKB and PDK1 proteins (Wild, Mut, or ΔPH) were expressed in 293 cells. Cells were serum starved in 0.2% (wt/vol) (white bars) or 0.1% (wt/vol) (dashed bars) BSA and stimulated for 5 min with insulin (10−6 M) (black bars), and PKB kinase activity was determined as described in Materials and Methods. Kinase activity is expressed as fold increase compared to that of nonstimulated wild-type cells. The levels of the expressed enzymes measured by immunoblotting are shown below the bar graph. (B) ΔPH-PKB and PDK1 constructs (Wild, Mut, or ΔPH) were expressed in 293 cells. Prior to stimulation for 5 min with insulin (10−6 M) (black bars) cells were serum starved in 0.2% (wt/vol) BSA, and ΔPH-PKB kinase activity was determined as described in Materials and Methods. Kinase activity is expressed as fold increase compared to that of ΔPH-PKB nonstimulated cells. Values shown are representative of at least three independent experiments performed in triplicate. The levels of the expressed enzymes measured by immunoblotting are shown below the bar graph. (C) 293 cells were transfected with ΔPH-PKB and with PDK1 and PH-PDK1 as indicated. ΔPH-PKB activity was determined as described in Materials and Methods. The levels of the expressed enzymes measured by immunoblotting are shown below the bar graph.
FIG. 7
FIG. 7
Working hypothesis for activation of PKB by PDK1. (A and B) Activation without insulin; (C and D) activation with insulin; (E and F) activation with or without insulin.
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