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1.
FIG. 2.

FIG. 2. From: Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor .

PTK6 colocalizes with AKT in transfected HEK-293 cells. Intracellular localization of AKT, mAKT and PTK6 was examined by indirect immunofluorescence in HEK-293 cells coexpressing PTK6-YF and AKT or mAKT. Myc-tagged PTK6 immunoreactivity is visualized with FITC (green), while HA-tagged AKT and mAKT are detected with rhodamine (red). Cells were counterstained with DAPI (blue). Transfected PTK6 and AKT colocalize in the cytoplasm (left panels), whereas mAKT is localized at the membrane (right panels). Size bar denotes 10 μm.

Yu Zheng, et al. Mol Cell Biol. 2010 September;30(17):4280-4292.
2.
FIG. 5.

FIG. 5. From: Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor .

PTK6 induces Src-family independent phosphorylation of AKT on tyrosine residues. (A) Immunoblotting demonstrates the absence of Src expression in SYF cells. Wild-type MEFs express high levels of Src kinase. (B) Immunoblot analysis of the lysates from SYF cells cotransfected with PTK6-YF and AKT plasmids. Total cell lysates were analyzed using antiphosphotyrosine (PY), -AKT, -Myc tag, and -α-tubulin antibodies. (C) Immunoblot analysis of AKT immunoprecipitated (IP) from lysates of SYF cells cotransfected with PTK6-YF and AKT plasmids. Tyrosine phosphorylation of AKT was analyzed using anti-PY antibodies (arrow). The membrane was reprobed with an antibody directed against AKT.

Yu Zheng, et al. Mol Cell Biol. 2010 September;30(17):4280-4292.
3.
FIG. 8.

FIG. 8. From: Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor .

Knockdown of PTK6 in BPH-1 cells impairs AKT activation in response to EGF. (A) BPH-1 cells stably expressing PLKO-1 vector (vec) or PTK6 shRNA were serum starved for 48 h and then stimulated with 0.1 ng/ml or 1 ng/ml EGF for 5 min. Cell lysates were analyzed by immunoblotting with anti-phospho-Thr-308, -phospho-Ser-473, -total AKT, -PTK6, -β-catenin, and -β-actin antibodies. β-Catenin and β-actin served as loading controls. (B) Immunoblot analysis of AKT immunoprecipitated (IP) from BPH-1 cell lysates previously described in the legend for panel A. Tyrosine phosphorylation of AKT was analyzed using anti-PY antibodies. The membrane was reprobed with an antibody directed against AKT.

Yu Zheng, et al. Mol Cell Biol. 2010 September;30(17):4280-4292.
4.
FIG. 1.

FIG. 1. From: Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor .

PTK6 induces phosphorylation of AKT on tyrosine residues. (A) Immunoblot analysis of total cell lysates of HEK-293 cells coexpressing increasing amounts (1, 2, and 3 μg) of Myc-PTK6-YF plasmid and 1 μg HA-AKT or HA-mAKT plasmid. Untreated (UN) HEK-293 cells served as a control. Total cell lysates were analyzed by immunoblotting with antiphosphotyrosine (PY), anti-PTK6, and anti-AKT antibodies. (B) Immunoblot analysis of AKT immunoprecipitated (IP) from lysates of HEK-293 cells coexpressing AKT or mAKT and PTK6-YF. AKT tyrosine phosphorylation was analyzed using anti-PY antibodies. Both short exposure (SE) and long exposures (LE) are shown. Arrows point at tyrosine-phosphorylated AKT and PTK6. The membrane was reprobed with antibodies directed against AKT and Myc-tagged PTK6. (C) Immunoblot analysis of phosphotyrosine-containing proteins that immunoprecipitated from lysates of HEK-293 cells coexpressing AKT or mAKT and PTK6-YF. The levels of AKT and PTK6 were analyzed using anti-AKT and anti-PTK6 antibodies.

Yu Zheng, et al. Mol Cell Biol. 2010 September;30(17):4280-4292.
5.
FIG. 4.

FIG. 4. From: Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor .

Interactions between PTK6 and AKT are mediated by the PTK6 SH2 and SH3 domains. (A) Immunoblot analysis of PTK6 immunoprecipitated (IP) from lysates of HEK-293 cells coexpressing PTK6-YF and wild-type AKT or AKT mutants (Y315F, Y326F, Y315F/Y326F and Y315E/Y326E). AKT association was analyzed using an anti-HA tag antibody. The membrane was stripped and reprobed with an antibody directed against Myc tag. Total cell lysates were analyzed by immunoblotting with anti-AKT, -PTK6, and -α-tubulin antibodies as input. (B) A column graph showing the ratio of AKT (HA-tag) to PTK6 (Myc-tag) band density quantified by NIH ImageJ software. (C) Immunoblot analysis of AKT immunoprecipitated (IP) from lysates of HEK-293 cells coexpressing PTK6-YF and wild-type AKT or AKT mutants (Y315F, Y326F, Y315F/Y326F, and Y315E/Y326E). The levels of tyrosine-phosphorylated AKT and PTK6 (pY-AKT and pY-PTK6) were analyzed using anti-PY antibodies (arrow). The level of PTK6 that interacts with AKT was analyzed using an anti-Myc tag antibody. The membrane was reprobed with an antibody directed against AKT. (D) GST pulldown assay using lysates of HEK-293 cells expressing AKT alone or in combination with PTK6-YF. Glutathione-Sepharose 4B beads, which bind purified GST tag, GST-PTK6, GST-SH2, GST-SH3, or GST-SH2/SH3 protein, were used to pull down AKT from HEK-293 lysates. Levels of bound AKT were analyzed using an anti-AKT antibody. Ten percent of the lysates added to the pulldown reaction served as input. (E) Immunoblot analysis of HA tag IP from lysates of HEK-293 cells coexpressing GFP-PTK6 and wild-type AKT or AKT mutants (P424A/P427A and P467A/P470A). PTK6 association was analyzed using an anti-GFP antibody. The membrane was stripped and reprobed with an antibody directed against HA tag. Total cell lysates were analyzed by immunoblotting with anti-GFP, -HA tag, and -β-actin antibodies as input.

Yu Zheng, et al. Mol Cell Biol. 2010 September;30(17):4280-4292.
6.
FIG. 7.

FIG. 7. From: Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor .

Active PTK6 promotes AKT activation through phosphorylation on tyrosine residues 315 and 326. (A) HEK-293 and SYF cells were transiently transfected with PTK6-YF or empty vector for 24 h, serum starved for another 24 h, and then stimulated with a low dose of EGF (0.1 ng/ml) for 5 min. Cell lysates were analyzed by immunoblotting with anti-PY, -Myc tag, and -β-catenin antibodies. β-Catenin served as a loading control. (B) Immunoblot analysis of AKT immunoprecipitated (IP) from lysates of HEK-293 cells and SYF cells previously described in the legend for panel A. Tyrosine phosphorylation of AKT was analyzed using anti-PY antibodies. The membrane was reprobed with an antibody directed against AKT. (C) SYF cell lysates described in the legend for panel A were analyzed by immunoblotting with anti-phospho-Thr-308, -phospho-Ser-473, -total AKT, -Myc tag, -β-catenin, and -β-actin antibodies. (D) Immunoblot analysis of HA tag immunoprecipitated (IP) from lysates of SYF cells coexpressing PTK6-YF and wild-type AKT or AKT Y315F/Y326F mutant. AKT activation was analyzed using anti-phospho-Thr-308 antibodies. Both short exposure (SE) and long exposures (LE) are shown. The membrane was reprobed with an antibody directed against the HA-tag. Relative band densities were quantified with NIH ImageJ software and are indicated under corresponding bands. Total cell lysates were analyzed by immunoblotting with anti-phospho-Thr-308, -HA tag, -Myc-tag, -β-catenin, and -β-actin antibodies as input. β-Catenin and β-actin served as loading controls.

Yu Zheng, et al. Mol Cell Biol. 2010 September;30(17):4280-4292.
7.
FIG. 6.

FIG. 6. From: Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor .

PTK6 positively regulates AKT activation stimulated by physiological levels of EGF in SYF cells. (A) Increased activating phosphorylation of AKT on Thr-308 and Ser-473 was detected in SYF cells expressing PTK6-YF following addition of 0.1 ng/ml EGF. SYF cells stably expressing PTK6-YF or empty vector (vec) were serum starved for 48 h and then stimulated with different concentrations of EGF (0.1 ng/ml, 1 ng/ml, 10 ng/ml) for 5 or 15 min. Cell lysates were analyzed by immunoblotting with anti-AKT phospho-Thr-308, -AKT phospho-Ser-473, -AKT, -mouse PTK6, and -β-actin antibodies. (B) SYF cells stably expressing PTK6-YF or empty vector were serum starved for 48 h and then stimulated by 0.1 ng/ml EGF for 5 min. Samples were in triplicate. Cell lysates were analyzed by immunoblotting with anti-AKT phospho-Thr-308, -AKT -phospho-Ser-473, -AKT, -mouse PTK6, -phospho-p42/p44, and -α-tubulin antibodies. (C) The ratio of AKT Thr-308 phosphorylation and Ser-473 phosphorylation band density to total AKT band density was quantified with NIH ImageJ software. Results were formatted as means ± standard deviations of results from three independent experiments. *, P = 0.031; **, P = 0.0007. (D) Immunoblot analysis of AKT immunoprecipitated (IP) from SYF cell lysates described in the legend for panel B. Tyrosine phosphorylation of AKT was analyzed using anti-PY antibodies. (E) SYF cells stably expressing PTK6-YF, PTK6-KM, PTK6-WT, or empty vector were serum starved for 48 h and then stimulated with 0.1 ng/ml EGF for 5 min. Cell lysates were analyzed by immunoblotting with anti-AKT phospho-Thr-308, -AKT phospho-Ser-473, -AKT, -mouse PTK6, and -β-actin antibodies. Relative band densities were quantified with NIH ImageJ Software and are indicated under corresponding bands. (F) Growth curves of SYF cells stably expressing empty vector, PTK6-YF, PTK6-KM, or PTK6-WT.

Yu Zheng, et al. Mol Cell Biol. 2010 September;30(17):4280-4292.
8.
FIG. 3.

FIG. 3. From: Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor .

PTK6 directly phosphorylates AKT on tyrosine residues 315 and 326. (A) PTK6 directly phosphorylates AKT in an in vitro kinase reaction. Recombinant human PTK6 and human AKT were incubated in kinase buffer in the presence of ATP for 10 min at 30°C and then subjected to SDS-PAGE and immunoblot analysis with anti-PY, -AKT, and -PTK6 antibodies. (B) The MS/MS spectrum of the AKT peptide TFCGTPEYLAPEVLEDNDpYGR showing phosphorylation at Tyr326. The fragment ions at b9 and y11 minus phosphate prove that phosphorylation is present on the tyrosine residue on the C-terminal end of the peptide. (C) Immunoblot analysis of AKT immunoprecipitated (IP) from lysates of HEK-293 cells coexpressing PTK6-YF and wild-type AKT or AKT mutants (Y215F and Y315F/Y326F). AKT tyrosine phosphorylation was analyzed using anti-PY antibodies. Both short exposure (SE) and long exposures (LE) are shown. Arrows point at tyrosine-phosphorylated AKT and PTK6. The membrane was reprobed with an antibody directed against the HA tag. Total cell lysates were analyzed by immunoblotting with anti-AKT, -PTK6, and -α-tubulin antibodies as input. (D) Reduced phosphorylation of GST-AKT Y315F/Y326F in an in vitro kinase reaction. Recombinant human PTK6 and purified GST-tagged wild-type AKT or AKT Y315F/Y326F mutant were incubated in kinase buffer in the presence of ATP for 10 min at 30°C and subjected to SDS-PAGE and immunoblot analysis with anti-PY, -GST tag, and -PTK6 antibodies. (E) Schematic representation of tyrosine-phosphorylated AKT. Tyrosine residues 215 and 326 were identified as sites phosphorylated by PTK6 using tandem mass spectrometry (solid arrow). Tyrosine residue 315 was found to be targeted by PTK6 through point mutation studies (dashed arrow). Phosphorylation on tyrosine residue 474 was reported in reference 12.

Yu Zheng, et al. Mol Cell Biol. 2010 September;30(17):4280-4292.

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