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, 289 (14), 10045-56

Threonine 680 Phosphorylation of FLJ00018/PLEKHG2, a Rho Family-Specific Guanine Nucleotide Exchange Factor, by Epidermal Growth Factor Receptor Signaling Regulates Cell Morphology of Neuro-2a Cells

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Threonine 680 Phosphorylation of FLJ00018/PLEKHG2, a Rho Family-Specific Guanine Nucleotide Exchange Factor, by Epidermal Growth Factor Receptor Signaling Regulates Cell Morphology of Neuro-2a Cells

Katsuya Sato et al. J Biol Chem.

Abstract

FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. The function of FLJ00018 is regulated by the interaction of heterotrimeric GTP-binding protein Gβγ subunits or cytosolic actin. However, the details underlying the molecular mechanisms of FLJ00018 activation have yet to be elucidated. In the present study we show that FLJ00018 is phosphorylated and activated by β1-adrenergic receptor stimulation-induced EGF receptor (EGFR) transactivation in addition to Gβγ signaling. FLJ00018 is also phosphorylated and activated by direct EGFR stimulation. The phosphorylation of FLJ00018 by EGFR stimulation is mediated by the Ras/mitogen-activated protein kinase (MAPK) pathway. Through deletion and site-directed mutagenesis studies, we have identified Thr-680 as the major site of phosphorylation by EGFR stimulation. FLJ00018 T680A, in which the phosphorylation site is replaced by alanine, showed a limited response of the Neuro-2a cell morphology to EGF stimulation. Our results provide evidence that stimulation of the Ras/MAPK pathway by EGFR results in FLJ00018 phosphorylation at Thr-680, which in turn controls changes in cell shape.

Keywords: Epidermal Growth Factor Receptor (EGFR); Guanine Nucleotide Exchange Factor (GEF); Neurite Outgrowth; Protein phosphorylation; Rho GTPases.

Figures

FIGURE 1.
FIGURE 1.
Effect of isoproterenol on FLJ00018-induced SRE-dependent gene transcription in β1-AR-expressing NIH3T3 cells. A, NIH3T3 cells were co-transfected with pSRE.L-luciferase, pRL-SV40 plasmid DNAs, and expression vectors for FLJ00018 (F018), β1-AR, and β-ARKct as indicated. Transfected cells were stimulated with 20 μm isoproterenol for 5.5 h. Luciferase activity was determined by a dual-luciferase reporter assay. Luciferase activity obtained with mock cells was taken as 1.0, and relative activities are shown. Values represent the means ± S.D. from at least three experiments. B, NIH3T3 cells were co-transfected with expression vectors for β1-AR and FLJ00018 as indicated. Transfected cells were stimulated with isoproterenol (20 μm) for 15 min. Equal amounts of protein were resolved by 7.5% SDS-PAGE. Immunoblotting (IB) was performed with antibodies against Myc.
FIGURE 2.
FIGURE 2.
FLJ00018 is activated by β1-AR-mediated transactivation of EGF receptors. A, NIH3T3 cells were co-transfected with expression vectors for EGFR, β1-AR, and FLJ00018 (F018) as indicated. Transfected cells were stimulated with isoproterenol (20 μm) for 0–30 min. Equal amounts of protein were resolved by 7.5% SDS-PAGE. Immunoblotting (IB) was performed with antibodies against phosphotyrosine (P-Tyr) or EGFR. B, NIH3T3 cells were co-transfected with pSRE.L-luciferase, pRL-SV40 plasmid DNAs, and expression vectors for FLJ00018, β1-AR, and EGFR as indicated. Transfected cells were stimulated with 20 μm isoproterenol for 5.5 h before treatment with 10 μm AG1478 for 1 h. Luciferase activity was determined by a dual-luciferase reporter assay. Luciferase activity obtained with mock cells was taken as 1.0, and relative activities are shown. Values are the means ± S.D. from at least three experiments. #, p < 0.05. C, NIH3T3 cells were co-transfected with expression vectors for EGFR, β1-AR, Myc-tagged FLJ00018, and Gβ1γ2 as indicated. Transfected cells were stimulated with isoproterenol (20 μm) for 15 min. Equal amounts of protein were resolved by 7.5% SDS-PAGE. To detect Myc-tagged FLJ00018, immunoblotting was performed with antibodies against Myc (Myc). D, NIH3T3 cells were co-transfected with expression vectors for EGFR, β1-AR, and Myc-tagged FLJ00018 as indicated. Transfected cells were stimulated with isoproterenol (20 μm) for 15 min. After lysis, the cell lysates were incubated with alkaline phosphatase (AP; 10 units) and λ-phosphatase (λ; 400 units) for 2 h at 30 °C. The reaction was terminated by the addition of SDS sample buffer. Equal amounts of the reaction mixture were resolved by 7.5% SDS-polyacrylamide gel electrophoresis. To detect Myc-tagged FLJ00018, immunoblotting was performed with antibodies against Myc (Myc). E and G, HEK293 cells (E) or NIH3T3 cells (G) were co-transfected with expression vectors for EGFR, β1-AR, and Myc-tagged FLJ00018. Transfected cells were treated with 10 μm AG1478 for 30 min (G) before stimulation with 20 μm isoproterenol for 15 min. Equal amounts of protein were resolved by 7.5% SDS-PAGE (E and G) or 3% SDS-polyacrylamide gel containing 13 μm Phos-tag, 26 μm MnCl2 and 1.5% agarose (F). Immunoblotting was performed with anti-Myc.
FIGURE 3.
FIGURE 3.
FLJ00018 is activated by EGF receptor signaling. A and B, NIH3T3 cells were co-transfected with pSRE.L-luciferase, pRL-SV40 plasmid DNAs, and expression vectors for FLJ00018 (F018), EGFR, RhoA T19N, Rac T17N, and Cdc42 T17N as indicated. Transfected cells were stimulated with 20 ng/ml EGF for 6 h. Luciferase activity was determined by a dual-luciferase reporter assay. Luciferase activity obtained with mock cells was taken as 1.0, and relative activities are shown. Values are the means ± S.D. from at least three experiments. C–G, NIH3T3 cells (C, D, and G) or HEK293 cells (E and F) were co-transfected with expression vectors for Myc-tagged FLJ00018, EGFR, and/or Gβ1γ2 as indicated. Transfected cells were stimulated with 20 ng/ml EGF for 15 min (C and E) or 0–30 min as indicated (G). After lysis, the cell lysates were incubated with alkaline phosphatase (AP; 10 units) and λ-phosphatase (λ; 400 units) for 2 h at 30 °C, and the reaction was terminated by the addition of SDS sample buffer (D). IB, immunoblot. Equal amounts of protein were resolved by 7.5% SDS-polyacrylamide gel electrophoresis (C–E and G) or by 3% SDS-polyacrylamide gel containing 13 μm Phos-tag, 26 μm MnCl2, and 1.5% agarose (F). To detect Myc-tagged FLJ00018, immunoblotting was performed with antibodies against Myc. H, NIH3T3 cells were co-transfected with expression vectors for Halo-tagged FLJ00018 or Halo-tagged Vav3 and EGFR as indicated. Transfected cells were stimulated with 20 ng/ml EGF for 15 min. Equal amounts of protein were resolved by 7.5% SDS-PAGE. To detect Halo-tagged FLJ00018 or Vav3, immunoblotting was performed with antibodies against Halo. Tyrosine phosphorylation of proteins was detected by antibodies against phosphotyrosine (P-Tyr). Dotted triangle, FLJ00018; open triangles, Vav3; closed triangles, EGFR tyrosine phosphorylation.
FIGURE 4.
FIGURE 4.
FLJ00018 is activated through H-Ras. A and B, NIH3T3 cells were co-transfected with expression vectors for FLJ00018 (F018), EGFR, and a constitutively active form of H-Ras (H-RasCA) (A) or a dominant-negative form of H-Ras (H-RasDN) (B) as indicated. Transfected cells were stimulated with 20 ng/ml EGF for 15 min. Equal amounts of the proteins were resolved by 7.5% SDS-PAGE. To detect Myc-tagged FLJ00018, immunoblotting was performed with antibodies against Myc.
FIGURE 5.
FIGURE 5.
FLJ00018 is activated through Ras/MAPK pathway. A and D, NIH3T3 cells were co-transfected with expression vectors for FLJ00018 (F018) and EGFR as indicated. Transfected cells were treated with 10 μm KN-93 (A), 10 μm U-0126 (A), 10 μm SB202190 (SB) (D), or 10 μm SP600125 (SP) (D) for 30 min before stimulation with 20 ng/ml EGF for 15 min. Equal amounts of proteins were resolved by 7.5% SDS-PAGE. To detect Myc-tagged FLJ00018, immunoblotting (IB) was performed with antibodies against Myc. B, C, and E, NIH3T3 cells were co-transfected with pSRE.L-luciferase, pRL-SV40 plasmid DNAs, and expression vectors for FLJ00018 and EGFR as indicated. Transfected cells were treated with KN-93 (10 μm) (B), U-0126 (10 μm) (C), SB202190 (SB) (10 μm) (E), or SP600125 (SP) (10 μm) (E) for 1 h before stimulation with 20 ng/ml EGF for 6 h. Luciferase activity was determined by dual-luciferase reporter assay. Luciferase activity obtained with mock cells was taken as 1.0, and relative activities are shown. Values are the means ± S.D. from at least three experiments.
FIGURE 6.
FIGURE 6.
Identification of the structure involved in FLJ00018 phosphorylation by EGFR signaling. A, structure of FLJ00018 mutants. The WT, P1, P1.5, P2, P3, and ΔN constructs code for amino acid (aa) residues 1–1386, 1–970, 1–615, 1–464, 1–310, and 965–1386 of FLJ00018, respectively. B and C, NIH3T3 cells were co-transfected with expression vectors for EGFR and Myc-tagged FLJ00018 mutants as indicated. Transfected cells were stimulated with 20 ng/ml EGF for 15 min. Equal amounts of proteins were resolved by 7.5% or 10% SDS-PAGE (B) or 6% SDS-polyacrylamide gel containing 15 μm Phos-tag and 30 μm MnCl2 (C). Immunoblotting (IB) was performed with antibodies against Myc. Closed triangles, shifted bands; Open triangles, basal levels of proteins.
FIGURE 7.
FIGURE 7.
FLJ00018 is phosphorylated by EGFR at amino acid residue threonine 680. A and B, NIH3T3 cells (A) or HEK293 cells (B and C) were co-transfected with expression vectors for EGFR and the Myc-tagged FLJ00018 or FLJ00018 T680A mutant as indicated. Transfected cells were stimulated with 20 ng/ml EGF for 15 min. Equal amounts of proteins were resolved by 7.5% (A and B) or 3% SDS-polyacrylamide gel containing 13 μm Phos-tag, 26 μm MnCl2, and 1.5% agarose (C). Immunoblotting (IB) was performed with antibodies against Myc). D and E, NIH3T3 cells were co-transfected with pSRE.L-luciferase, pRL-SV40 plasmid DNAs, expression vectors for FLJ00018 (F018), and EGFR, RhoA T19N, Rac T17N, and Cdc42 T17N as indicated. Transfected cells were stimulated with 20 ng/ml EGF for 6 h. Luciferase activity was determined by a dual-luciferase reporter assay. Luciferase activity obtained with mock cells was taken as 1.0, and relative activities are shown. Values are the means ± S.D. from at least three experiments. WT, FLJ00018 WT; TA, FLJ00018 T680A mutant.
FIGURE 8.
FIGURE 8.
FLJ00018 activation causes neurite outgrowth via EGFR activation. A, Neuro-2a cells were transfected with expression vectors for EGFR and the mAG-tagged FLJ00018 (F018) or FLJ00018 T680A mutant as indicated. Transfected cells were stimulated with 20 ng/ml EGF for 24 h. After stimulation, cells were fixed by 4% paraformaldehyde. Fluorescence images were acquired using a fluorescence microscope. Scale bar, 20 μm. B, 10 fluorescence images were taken from random fields and analyzed using Image J software. The total neurite length of 50 cells was measured in each sample. Total neurite lengths were analyzed using Image J software. Values are the means ± S.D. from at least three experiments. #, p < 0.05.
FIGURE 9.
FIGURE 9.
Proposed mechanisms of FLJ00018 activation through EGFR transactivation. Stimulated β1-AR induces EGFR transactivation. Through the MAPK cascade, activated EGFR regulates and phosphorylates FLJ00018. FLJ00018 may be phosphorylated by MEK or its downstream effectors, and the phosphorylated FLJ00018 may in turn activate Rho family members Cdc42 and Rac. In Neuro-2a cells, phosphorylated FLJ00018 regulates neurite outgrowth.

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