Glycogen synthase kinase 3- and extracellular signal-regulated kinase-dependent phosphorylation of paxillin regulates cytoskeletal rearrangement

Abstract

Paxillin is a 68-kDa focal adhesion-associated protein that plays an important role in controlling cell spreading and migration. Phosphorylation of paxillin regulates its biological activity and thus has warranted investigation. Serine 126 and serine 130 were previously identified as two major extracellular signal-regulated kinase (ERK)-dependent phosphorylation sites in Raf-transformed fibroblasts. Here serine 126 is identified as a phosphorylation site induced by lipopolysaccharide (LPS) stimulation of RAW264.7 cells. A number of other stimuli, including adhesion and colony-stimulating factor, induce serine 126 phosphorylation in RAW264.7 cells, and nerve growth factor (NGF) treatment induces serine 126 phosphorylation in PC12 cells. The kinase responsible for phosphorylation of this site is identified as glycogen synthase kinase 3 (GSK-3). Interestingly, this GSK-3-dependent phosphorylation is regulated via an ERK-dependent priming mechanism, i.e., phosphorylation of serine 130. Phosphorylation of S126/S130 was required to promote spreading in paxillin null cells, and LPS-induced spreading of RAW264.7 cells was inhibited by expression of the paxillin S126A/S130A mutant. Furthermore, this mutant also retarded NGF-induced PC12 cell neurite outgrowth. Hence, phosphorylation of paxillin on serines 126 and 130, which is mediated by an ERK/GSK-3 dual-kinase mechanism, plays an important role in cytoskeletal rearrangement.

FIG. 1.

LPS induces paxillin tyrosine and serine phosphorylation. (A) RAW264.7 cells were serum starved overnight (lane 1) and then stimulated with 1 μg/ml LPS for 1 h (lanes 2, 3) prior to lysis. Paxillin was immunoprecipitated from 500 μg of cell lysate. Half of the immune complex from the LPS-stimulated sample was incubated with calf intestine alkaline phosphatase for half an hour (lane 3). The immune complexes were then analyzed by Western blotting. (B) Cell lysates from unstimulated (lane 1) and LPS-stimulated (lane 2) RAW264.7 cells were Western blotted using phospho-specific (top four panels) or paxillin (bottom panel) antibody. (C) RAW264.7 cell line derivatives stably expressing EGFP-paxillin (lanes 1, 2) or EGFP-paxillin S126A (lanes 3, 4) were serum starved (lanes 1, 3) or starved and then stimulated with LPS (lanes 2, 4). Cell lysates were blotted with the PS126 (top) or paxillin (bottom) antibody. The positions of the exogenous EGFP-paxillin and endogenous paxillin are indicated. (D) Primary peritoneal macrophages (lanes 1 to 6) were stimulated with LPS (1 μg/ml) for the indicated times prior to lysis. J774 cells (lanes 7, 8) and Bac1 cells (lanes 9, 10) were starved (lanes 7, 9) or stimulated with LPS (1 μg/ml) (lanes 8, 10) for 1 h prior to lysis. Lysates were blotted with PS126 or paxillin antibody. (E) Lysates from RAW264.7 cells stimulated with LPS were analyzed. Fifty micrograms of lysate was directly analyzed (lane 1) or 500 μg of lysate was immunoprecipitated using PS126. Ten percent of the immune complex (IP) (lane 2) or supernatant (lane 3) was analyzed by Western blotting for paxillin.

FIG. 2.

LPS-induced paxillin serine 126 phosphorylation is ERK dependent. (A) RAW264.7 cells were serum starved overnight (lane 1) and then stimulated with 1 μg/ml LPS for the indicated time (lanes 2-4). Cell lysates were blotted with the PS126, paxillin, pERK, and ERK2 antibodies. (B) RAW264.7 cells were serum starved overnight (lane 1) and then pretreated with the MEK inhibitor PD98095 at the dose shown for 1 h. Cells were stimulated with LPS (1 μg/ml) for 1 h. Cell lysates were blotted with PS126, paxillin, pERK, or ERK antibody. (C) RAW264.7 cells were treated as in panel B, except that MEK was inhibited with 20 μM U0126. (D) Wild-type or constitutively activated MEK was transiently expressed in 293 cells, and lysates were blotted with the PS126, paxillin, or pERK antibody.

FIG. 3.

Paxillin serine and tyrosine phosphorylation are regulated by separate pathways. (A) RAW264.7 cell line derivatives transiently expressing EGFP-paxillin (lanes 1, 2) or EGFP-paxillin Y31F/Y118F (lanes 3, 4) were stimulated with LPS (lanes 2, 4) and cell lysates blotted with the paxillin or PS126 antibody. The positions of the exogenous EGFP-paxillin and endogenous paxillin are indicated. (B) RAW264.7 cells were serum starved overnight (lane 1) and then pretreated with the Src inhibitor PP2 at the doses shown for 20 min. Cells were stimulated with LPS (1 μg/ml) for 1 h. Cell lysates were blotted with PS126, PY118, paxillin, pERK, or ERK antibody. (C) RAW264.7 cells were serum starved overnight (lane 1) and then were pretreated with U0126 at the dose shown for 1 h. Cells were stimulated with LPS (1 μg/ml) for 1 h. Cell lysates were blotted with the paxillin or PY118 antibody.

FIG. 4.

Paxillin serine 126 phosphorylation is induced by multiple stimuli. (A) RAW264.7 cells were trypsinized, incubated in suspension at 37°C for half an hour (lane 2), and then replated on fibronectin-coated plates (50 μg/ml) for half an hour (lane 3) at 37°C prior to lysis. Cell lysates were blotted with paxillin or PS126 antibody. (B and C) RAW264.7 cells were serum starved overnight (lane 1) and then were stimulated with PMA (100 ng/ml) (lane 2 in panel B), LPS (1 μg/ml) (lane 2 in panel C), or CSF (1.32 nM) (lane 3 in panel C) for 1 h. Cell lysates were blotted with paxillin, PS126, or pERK antibody. (D) PC12 cells were starved in DMEM containing 1% FBS for 6 h, treated with 20 μM U0126 for 1 h (lane 3), and stimulated with 100 ng/ml NGF for 2 h (lanes 2, 3). Cell lysates were blotted with PS126, paxillin, or pERK antibody.

FIG. 5.

Phosphorylation of serine 126 is abolished by GSK-3 inhibitors. (A, B, and C) RAW264.7 cells were serum starved overnight (lane 1) and then pretreated with LiCl, GSK-3 inhibitor IX, or GSK-3 inhibitor I at the dose indicated for 1 h. Cells were stimulated with LPS (1 μg/ml) for 1 h. Cell lysates were blotted with the indicated antibodies. (D) PC12 cells were starved in DMEM containing 1% FBS for 6 h, treated with the indicated drug for 1 h, and stimulated with 100 ng/ml NGF for 2 h. Cell lysates were blotted with the indicated antibodies. (E) RAW264.7 cells were transfected with the control or GSK-3β siRNAs, starved, and stimulated with LPS. Lysates were blotted for PS126, paxillin, pERK, or GSK-3α/β.

FIG. 6.

Phosphorylation of paxillin is mediated by ERK/GSK-3 dual-kinase mechanism. (A) Recombinant GST-N-C3 was incubated with active GSK-3 and ERK1, alone or in combination, in 50 μl of kinase reaction buffer at 30°C for 60 min. Samples were resolved by SDS-PAGE and immunoblotted using the PS126 or paxillin antibody. (B) GST-N1-C1A was incubated with ERK in kinase reaction buffer containing [γ-³²P]ATP and the samples resolved by SDS-PAGE. The autoradiograph and Coomassie-stained gel are shown. (C) The wild-type GST-N-C3 or GST-N-C3 variant containing an alanine substitution for serine 130 was treated as for panel A. (D) PC12 cells transiently expressing EGFP-paxillin (lanes 1, 2) or EGFP-paxillin S130A (lanes 3, 4) were challenged with NGF (lanes 2, 4), and cell lysates were blotted with the paxillin or PS126 antibody. The positions of the exogenous EGFP-paxillin and endogenous paxillin are shown. (E) A model for phosphorylation of paxillin by the ERK/GSK-3 dual-kinase mechanism is shown.

FIG. 7.

Subcellular localization of the serine 126-phosphorylated form of paxillin in fibroblasts. (A) Paxillin null fibroblasts were transiently transfected with EGFP-paxillin (top panels) or the EGFP-paxillin S126A mutant (bottom panels). Twenty-four hours later, the cells were trypsinized, held in suspension for 45 min and then plated onto fibronectin-coated coverslips for 60 min in serum-free medium. Localization of the protein was visualized by fluorescence microscopy (left panels). Localization of paxillin that was phosphorylated on serine 126 was determined by immunofluorescence using the PS126 antibody (right panels). (B) PC12 cells were serum starved (left panel) and stimulated with NGF (100 ng/ml) for 3 h (middle panel) or 30 h (right panel). Cells were fixed and immunostained using the PS126 antibody.

FIG. 8.

ERK/GSK-3-mediated phosphorylation of paxillin is involved in cell spreading. (A) Lysates from paxillin null cells or cells reexpressing wild-type or S126A/S130A paxillin were analyzed by Western blotting for paxillin (top panel) or PS126 (bottom panel). (B) Cells were serum starved overnight and then plated on fibronectin. At various times, the area of spreading cells was determined using Image J software (>50 cells per experiment) and the average from three experiments plotted. The data at each time point were analyzed using one-way analysis of variance (P < 0.0001 at each time) and the Dunnett post test (for wild-type-expressing cells, P < 0.01 at each time; for S126A/S130A cells, P < 0.05 only at 60 min). (C) RAW264.7 cells or derivatives stably expressing EGFP-paxillin or EGFP-paxillin S126A/S130A were challenged with 1 μg/ml LPS as described. Cell spreading was recorded after 3 h of LPS stimulation. Phase-contrast images (left panels) and fluorescent images (right panels) of cells transfected with EGFP-paxillin (top panels) or EGFP-paxillin S126A/S130A (bottom panels) are shown. (D) GFP-negative, untransfected cells and GFP-positive cells expressing exogenous paxillin were scored as spread or unspread, and percentages of cells spread were analyzed. Greater than 100 GFP-positive cells were scored in each experiment. The data were plotted as the means ± standard errors from three experiments. The P value is less than 0.05 (unpaired student t test).

FIG. 9.

ERK/GSK-3-mediated phosphorylation of paxillin is involve in NGF-induced neurite outgrowth. (A) PC12 cells transiently expressing EGFP-paxillin or EGFP-paxillin S126A/S130A were plated on collagen-coated petri dishes and treated with 100 ng/ml NGF. Neurite outgrowth was recorded after 36 h of NGF stimulation. Phase-contrast images (left panels) and fluorescent images (right panels) of cells transfected with EGFP-paxillin (top panels) or EGFP-paxillin S126A/S130A (bottom panels) are shown. (B) Quantitative analysis of neurite outgrowth from three independent experiments is shown. In each experiment, more than 100 GFP-negative, untransfected cells (light-gray bars) and GFP-positive cells from the EGFP-paxillin (white bars), EGFP-paxillin S126A/S130A (black bars), or EGFP-paxillin S126D/S130D (dark gray bars) transfected populations were scored. The data was plotted as the means ± standard errors from three experiments. In each group the wild-type and S126A/S130A data were analyzed using one-way analysis of variance and the Dunnett post test (for each: ANOVA, P < 0.0002; Dunnett, P < 0.01).