Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity

Abstract

The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration.

Fig. 1

WAVE2 undergoes a mobility shift assessed by SDS-PAGE. (A) NIH 3T3 fibroblasts were serum starved and stimulated with 10% serum for the indicated times. Cells were incubated with 10 μM Uo126 or DMSO control where indicated. Lysates were immunoblotted for the indicated protein species. ErkP, phospho-Erk; Erk t, total Erk. (B) Domain structure of WAVE2 and truncation constructs. (C) GST-WAVE2 constructs were purified from Cos-7 cells growing in 10% serum and treated with alkaline phosphatase (AP) where indicated before resolution by SDS-PAGE and silver staining. (D) GST-∆SHD/∆VCA constructs containing the indicated alanine (A) mutations were purified and treated as above. 308A is included as an example of a Ser-to-Ala mutation that does not affect the mobility of WAVE2. Note the disappearance of faint upper species in the 343A mutant. An aliquot of each phosphatase-treated sample was incubated with recombinant Erk2 (E). AAA, 343A-346A-351A triple mutant.

Fig. 2

Phosphorylation of WAVE2 by Erk and Jnk. (A) GST-∆SHD/∆VCA constructs were purified and treated as described in Fig. 1 before immunoblotting with antibodies raised against the indicated phospho-peptides. WT, wild type. (B) FLAG-tagged full-length WAVE2 was expressed in, and immunoprecipitated from, Cos-7 cells and either untreated (−), phosphatase treated (AP) or phosphatase treated and then incubated with recombinant Erk2 or Jnk2 as indicated. Samples were analysed by immuno-blotting with the indicated antibodies. (C) Serum-starved NIH 3T3 fibroblasts were stimulated with PDGF-ββ for the indicated times, before lysis. Immunoprecipitated WAVE2 was immunoblotted as indicated. Lysates were immunoblotted with antibodies against phospho-Erk (ErkP) to monitor activation of the Erk pathway. (D) Serum-starved NIH 3T3 cells were stimulated with PDGF-ββ for the indicated times, lysed and WAVE2 phosphorylation monitored by phospho-specific immunoblotting. Densitometry of phospho-WAVE2 signals compared to total WAVE2 signal after stripping was averaged for three separate experiments. (E) NIH 3T3 cells were preincubated with the indicated inhibitors [Uo126 at 10 μM (Uo), SP600125 at 10 μM (SP), SB202190 at 10 μM (SB), SB203580 at 10 μM (SB’), wortmannin at 100 nM (W)] before stimulation with PDGF for 30 minutes, lysis and immunoprecipitation of WAVE2. Lysates were immunoblotted with antibodies against ErkP to assess inhibition of this pathway. The blots shown are representative of numerous experiments.

Fig. 3

Phospho-defective WAVE2 expression does not affect ruffling in response to PDGF. (A) NIH 3T3 cells stably expressing EGFP, EGFP-WAVE2-wild-type (W2 WT) or EGFP-WAVE2-AAA (W2 AAA) were plated on fibronectin-coated glass coverslips overnight in 0.5% serum. PDGF was added where indicated for 25 minutes before cells were fixed, permeabilised and stained with phalloidin (F-actin). Bar, 30 μm. (B) Experiments were performed as for A, then six random microscopic fields were imaged and the number of cells with one or more ruffles were counted. Dark bars (+) represent cells with ruffles, and light-grey bars (−) represent those without ruffles. Statistical analysis with Fisher’s exact test revealed no significant differences between cell lines in either starved or stimulated conditions.

Fig. 4

WAVE2 phosphorylation following scratch-wounding. (A) Native NIH 3T3 cells (left panels) or NIH 3T3 cells expressing EGFP-WAVE2-wild-type (right panels) were grown to confluence and scratched. Cultures were left for the indicated times before lysis and immunoprecipitation of endogenous WAVE2 or EGFP-WAVE2 with the indicated antibodies. Immunoprecipitates were blotted as shown. Lysates were taken and blotted for phospho-Erk and phospho-Jnk. (B) In addition to the immunoprecipitates shown in A, NIH 3T3 cells expressing EGFP-WAVE2-AAA were also wounded. Immunoprecipitates were resolved next to a sample of lysate (w) and immunoblotted as indicated.

Fig. 5

Phosphorylation of WAVE2 is required to maintain the directional persistence of cell migration. (A) NIH 3T3 cells stably expressing EGFP, EGFP-WAVE2-wild-type (WT) or EGFP-WAVE2-AAA (AAA) were treated with control siRNA (C) and grown to confluence in a chamber slide. Monolayers were scratch-wounded and cell migration was filmed using time-lapse microscopy. Frames were taken from a representative movie (see supplementary material Movies 1-3 for originals) showing migration at the indicated time points. (B) Cells were tracked every 30 minutes from 8-16 hours post-wounding and their paths overlayed. Paths are oriented such that the start point is normalised to the origin and the wound face runs parallel to the x-axis. All tracks from several fields in this representative experiment are shown. (C,D) Experiment performed as for A and B except that cells were treated with WAVE2 siRNA 48 hours before wounding (see supplementary material Movies 4-6 for originals). (E) Cells from the experiment described in panels A-D were plated in parallel and lysates immunoblotted as indicated.

Fig. 6

WAVE2 regulates polarisation of the Golgi apparatus. (A) Histogram showing the number of NIH 3T3 cells with polarised Golgi apparatus following treatment with siRNA targeting either: nothing (cont), WAVE1 (W1), WAVE2 (W2) or both. Data from three separate experiments were collated. *** indicates a P value <0.0001 (Fisher’s exact test) for the null hypothesis that there is no difference between WAVE- and control-knockdown cells. Percentage figures indicate the percentage of cells with a polarised Golgi apparatus. P, polarised Golgi; NP, nonpolarised Golgi. (B) Typical immunoblot analysis of protein levels following treatment with siRNA. (C) Visualisation of the Golgi apparatus (antibody against GM130) in green, F-actin (phalloidin) in red and nuclei (DAPI) in blue following control siRNA (cont) or WAVE2 siRNA. The wound edge is oriented vertically at the right of each panel. The lower panel shows GM-130 staining alone. Bar, 20 μm.

Fig. 7

Phosphorylation of WAVE2 regulates polarisation of the Golgi apparatus. (A) Sample images taken 3 hours post wounding of cells expressing the indicated transgene (EGFP-WT refers to wild-type WAVE2 and EGFP-AAA refers to the triple alanine mutant of WAVE2). EGFP + KD indicates EGFP control cells treated with WAVE2 siRNA. The Golgi apparatus was visualised with an antibody against GM130, and F-actin with phalloidin. Bar, 20 μm. (B) The polarisation of the Golgi apparatus was quantified in cells overexpressing either EGFP (GFP), EFGP-WAVE2-WT (GFP-WT): P values derived by Fisher’s exact test are indicated below the percentage figures indicating the percentage of cells with a normally polarised Golgi apparatus. (C) Visualisation of disruption of the Golgi apparatus following treatment with Uo126 and SP600125, each at 10 μM (Uo126+SP). (D) Quantitation of the effects of 10 μM Uo126 (Uo) and SP600125 (SP) on polarisation of the Golgi apparatus. Cell number refers to wound-edge cells with a polarised Golgi apparatus. *** indicates a P value <0.0001 for inhibitor-treated cells compared with DMSO-treated controls. P, polarised Golgi; NP, nonpolarised Golgi.