Results: 4

1.
Figure 3

Figure 3. From: Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton.

Activation of PKN by PDK1. (A) Activation of PKN by PDK1 in vitro. The FLAG-tagged wild-type PKN or PKNT774A was immunoprecipitated from serum-starved CHO/IR cells transiently expressing these proteins, phosphorylated in vitro in the presence (+) or absence (−) of affinity-purified PDK1, and assayed for phosphorylation of the S6 peptide. Data are representative of three independent experiments, with bars representing means of duplicate determinations. (B) Activation of PKN by PDK1 in cells. CHO/IR cells were cotransfected with cDNAs encoding PKN, pcDNA/PDK1, or pcDNA/PDK1K114G and the control vector pcDNA. Twenty-four hours after transfection, cells were lysed and activity of the immunoprecipitated PKN was assayed as described in Materials and Methods. Data are mean ± SD of two independent experiments with duplicate determinations.

Lily Q. Dong, et al. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5089-5094.
2.
Figure 4

Figure 4. From: Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton.

Effects of PDK1 and PKN on actin cytoskeleton reorganization. (A) Ectopic expression of PDK1 and PKN induces actin cytoskeleton reorganization. 3T3-L1 (Upper) or Rat1-IR (Lower) cells were transfected with plasmids encoding FLAG-PKN, myc-PDK1, or myc-PKCζ, respectively. Arrows indicate cells expressing the recombinant proteins as determined by staining with antibodies to the tags. Actin cytoskeleton was detected by a rhodamine-phalloidin stain. (B) Ectopic expression of mutant PKN or PDK1 blocks insulin-induced actin cytoskeleton reorganization. Rat1-IR cells (a and e) or cells transiently expressing FLAG-PKNT774A (b and f), myc-PDK1K114G (c and g), or FLAG-PKNK644D (d and h) were serum starved for 4 h and treated with 10 nM insulin for 10 min (bh). Actin cytoskeleton was detected by a rhodamine-phalloidin stain (ae). The expression of the recombinant proteins in cells was determined by staining with the anti-FLAG or anti-myc antibodies, followed by Alexa Fluor 488-conjugated goat anti-mouse Ig (fh). Images are representative of those in three independent experiments. (C) Overexpression of PKNT774A inhibits PDK1-induced actin stress fiber reorganization. Rat1-IR cells were cotransfected with FLAG-tagged PKNT774A and myc-tagged PDK1. The expression of the recombinant proteins in cells was determined by staining with the anti-FLAG or anti-myc antibodies, followed by Alexa Fluor 488-conjugated goat anti-mouse Ig (green, for PKNT774A) or Alexa Fluor 350-conjugated goat anti-rabbit Ig (blue, for PDK1), respectively. Actin cytoskeleton was detected by a rhodamine-phalloidin stain (red). Results are representative of two independent experiments in which the inhibition of PDK1-induced actin stress fiber reorganization was observed in approximately 65% of cells coexpressing PKNT774A.

Lily Q. Dong, et al. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5089-5094.
3.
Figure 1

Figure 1. From: Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton.

Interaction of PKN with PDK1. (A) Schematic representation of PKN constructs used to study PKN and PDK1 interaction and phosphorylation. The consensus PDK1 phosphorylation sequences of several PDK1 substrates are shown. (B) Interaction of PDK1 with PKN in the yeast two-hybrid system. cDNAs containing different regions of PDK1 were amplified by PCR and subcloned into the yeast two-hybrid plasmid pGBT9. The interaction between PDK1 fragments and PKN-CT in SFY526 yeast cells was detected by β-galactosidase filter assays as described (14). +, Positive interaction (blue color) was visualized within 30 min; −, no interaction was detected for 24 h. N, Amino terminus; CD, catalytic domain; PH, pleckstrin homology domain. (C) Interaction between PDK1 and PKN in vitro. Lysates of CHO/IR/PDK1 cells (11) were incubated with GST/AF3-PKNK644E (lane 1) or GST/PKN(ΔC) (lane 2) bound to glutathione-Sepharose beads (Sigma). The bound proteins were resolved by SDS/PAGE, transferred onto a nitrocellulose membrane, and examined by immunoblotting with antibody to the hemagglutinin tag (Top). Five percent of cell lysates used in the immunoprecipitation were loaded as a control (Middle). The GST fusion proteins used in the experiments were separated by SDS/PAGE and visualized by Coomassie blue staining (Bottom). Results are representative of three independent experiments. (D) Interaction of PDK1 and PKN in intact cells. CHO/IR cells were serum-starved overnight and treated with or without wortmannin (WT, 200 nM) or LY294002 (LY, 200 μM) for 1 h. Cells then were treated with (+) or without (−) 10 nM insulin for 10 min. Cell lysates were incubated for 8 h at 4°C with affinity-purified polyclonal antibody to PDK1 (11) or NIg bound to protein A beads. PKN coimmunoprecipitated with PDK1 was detected with a mAb to PKN (Transduction Laboratories).

Lily Q. Dong, et al. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5089-5094.
4.
Figure 2

Figure 2. From: Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton.

Phosphorylation and activation of PKN by PDK1. (A) Time course study of PKN phosphorylation. The FLAG-tagged full-length PKN was immunoprecipitated from CHO/IR/PKN cells with the M2 anti-FLAG antibody. In vitro phosphorylation was carried out in the presence (+) or absence (−) of immunoaffinity-purified PDK1 (11). Reactions were stopped at indicated times. The phosphorylated proteins were eluted from beads by boiling in SDS-sample buffer, separated by SDS/PAGE, transferred to a nitrocellulose membrane, and visualized by autoradiography (Upper). The same membrane was reblotted with anti-FLAG antibody to ensure equal loading of proteins in each lane (Lower). (B) Phosphorylation of AF3-PKN by PDK1. AF3-PKN (15) was transiently expressed in CHO/IR cells, immunoprecipitated by anti-FLAG antibody, and phosphorylated in vitro in the presence of immunoaffinity-purified PDK1 or PDK1K114G, according to a protocol similar to that described previously (11). The phosphorylation of AF3-PKN was visualized by autoradiography. (C) Phosphoamino acid analysis of AF3-PKN. The 32P-labeled wild-type (WT) or T774A mutant of AF3-PKN bands were excised from the membrane, hydrolyzed, and analyzed by two-dimensional thin-layer electrophoresis, according to a protocol described previously (16). (D) The effect of PDK1 on PKN phosphorylation in cells. CHO/IR cells were transfected with PKN and PDK1, in vivo-labeled with 32P-orthophosphate, and treated with (+) or without (−) insulin as described in Materials and Methods. Data are mean ± SEM from four independent experiments. Background values obtained from NIg precipitates were subtracted from all values. (E) Overexpression of PDK1 stimulates PKN phosphorylation at Thr774 in intact cells. CHO/IR cells were cotransfected with FLAG-tagged PKN or PKNT774A and either myc-tagged PDK1 or PDK1K114G as indicated. Cell lysates were immunoprecipitated with antibody to the FLAG tag. One-eighth of the immunoprecipitates were separated by SDS/PAGE and analyzed by Western blot with antibody specific to phospho-Thr816 of PRK2 (Top). Two-thirds of the immunoprecipitates were used for Western blot with antibody to the FLAG tag (Middle). Expression of PDK1 in these cells was detected by Western blot of cell lysate (1/20 of total) using antibody to the myc tag (Bottom). (F) Insulin stimulates the phosphorylation of endogenous PKN at Thr774 in CHO/IR cells. CHO/IR cells were serum-starved overnight and treated with or without wortmannin (WT, 200 nM) or LY294002 (LY, 200 μM) for 1 h. Cells then were treated with (+) or without (−) 10 nM insulin for 10 min. Cell lysates were incubated for 8 h at 4°C with mAb to PKN (Transduction Laboratories) or NIg bound to protein G beads. One-eighth of the immunoprecipitates were separated by SDS/PAGE and analyzed by Western blot with antibody specific to phospho-Thr816 of PRK2 (Upper). Two-thirds of the immunoprecipitates were used for Western blot with a mAb to PKN (Transduction Laboratories) (Lower).

Lily Q. Dong, et al. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5089-5094.

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