PAK1-mediated activation of ERK1/2 regulates lamellipodial dynamics - PubMed (original) (raw)

. 2008 Nov 15;121(Pt 22):3729-36.

doi: 10.1242/jcs.027680. Epub 2008 Oct 21.

Affiliations

PAK1-mediated activation of ERK1/2 regulates lamellipodial dynamics

Stephen D Smith et al. J Cell Sci. 2008.

Abstract

PAK1 is a member of the p21-activated kinase (PAK) family of serine/threonine kinases that are activated by the Rho GTPases Rac and Cdc42, and are implicated in regulating morphological polarity, cell migration and adhesion. Here we investigate the function of PAK1 in cell motility using macrophages derived from PAK1-null mice. We show that CSF1, a macrophage chemoattractant, transiently stimulates PAK1 and MAPK activation, and that MAPK activation is reduced in PAK1-/- macrophages. PAK1 regulates the dynamics of lamellipodium extension as cells spread in response to adhesion but is not essential for macrophage migration or chemotaxis towards CSF1. Following adhesion, PAK1-/- macrophages spread more rapidly and have more lamellipodia than wild-type cells; however, these lamellipodia were less stable than those in wild-type macrophages. ERK1/2 activity was reduced in PAK1-/- macrophages during adhesion, and inhibition of ERK1/2 activation in wild-type macrophages was sufficient to increase the spread area and mimic the lamellipodial dynamics of PAK1-/- macrophages. Together, these data indicate that PAK1 signals via ERK1/2 to regulate lamellipodial stability.

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Figures

Fig. 1

Fig. 1

PAK1 regulates CSF-1-induced MAPK activation but not macrophage differentiation or chemotaxis. A, lysates from Wt and PAK1−/− BMMs were immunoblotted for PAK1 and PAK2 using a group 1 specific polyclonal antibody (C19) or a PAK1-specific polyclonal antibody. β-actin was used as a loading control. B, Flow cytometry analysis of Wt and PAK1−/− BMMs surface F4/80 expression levels, detected using a FITC-F4/80 antibody. Background fluorescence levels were established with a FITC-IgG2b negative control antibody. C, Wt BMMs were stimulated with 33 ng/ml CSF-1, and lysates were immunoblotted for phospho-Thr423-PAK1 and β-actin as a loading control. D, Wt and PAK1−/− BMMs were stimulated with 33 ng/ml CSF-1 and lysates were immunoblotted for phospho-Ser473-Akt, phospho-Thr202/Tyr204-ERK1/2, phospho-Thr180/Tyr182-p38 and phospho-Ser298-MEK1/2 levels. β-actin was used as a loading control. Western blots are representative of three separate experiments. E, To investigate chemotaxis, 1×105 Wt or PAK1−/− BMMs were placed into the upper chamber of a Transwell with 33 ng/ml CSF-1 in the lower chamber. After 24 hours, cell migration was evaluated by determining the cell number in ten randomly selected fields. Results are the mean +/− s.e.m. of 3 experiments performed in triplicate.

Fig. 2

Fig. 2

PAK1 regulates macrophage spreading but not migration speed. A, Wt and PAK1−/− BMMs were seeded onto tissue culture plastic in growth medium. Upper panels, representative micrographs of migrating BMMs 8 hours after seeding. Bar denotes 100 μm; arrows indicate round, flat cells, arrowheads indicate lamellipodia on migrating cells. Lower panels, cell migration was followed by time-lapse microscopy for 8 hours. Mean migration speed at each time point was determined from cell tracks,.= 70 and 60 for Wt and PAK1−/− BMMs respectively. Results are representative of three separate experiments. B, Wt and PAK1−/− BMMs were allowed to adhere to uncoated glass coverslips in macrophage starve medium supplemented with 33 ng/ml CSF-1 for the indicated times. Cells were stained for F-actin, Bar = 10 μm. C, The images in B were analysed to quantify the spread area of Wt and PAK1−/− BMMs. Quantification is the mean +/− s.e.m. of three separate experiments, n ~ 50 cells/time-point/experiment. D, Quantification of Wt and PAK1−/− BMM spread area as determined in C, using images of cells 24 hours after adhesion. Data are the mean +/− s.e.m. of three separate experiments, n ~ 50 cells/experiment. E, PAK1−/− BMMs nucleofected with GFP (upper panels) or GFP-PAK1 (lower panels) were allowed to adhere to uncoated glass coverslips in growth medium for the indicated times. Cells were stained for F-actin, bar = 10 μm. The spread area of GFP- and GFP-PAK1-expressing cells was determined. Data are the mean +/− s.d. of two separate experiments, n ~ 20 cells/experiment.

Fig. 3

Fig. 3

PAK1 is required for lamellipodial stability during spreading. A, Wt and PAK1−/− BMMs were plated onto glass-bottomed culture dishes in growth medium and cell spreading was visualised by time-lapse microscopy. Images at the indicated time-points of the movies are shown. Bar = 1 μm. B, The length and the perimeter of Wt and PAK1−/− lamellipodia was determined using movies as in A. Data shown are the mean +/− s.e.m, n = 145 and 225 lamellipodia from Wt and PAK1−/− BMMs respectively, from 6 separate experiments. **p<0.01, ***p<0.001 PAK1−/− compared to Wt, Student’s _t_-test. C, Kymographs of extending lamellipodia. The final frame from the movie used (left panels) indicates the region of kymograph production. Right panels show kymographs, the inset shows the kymograph with the membrane edge highlighted (white line). D, Quantification of the number of lamellipodia/cell observed at the specified frames in movies. Mean +/− s.e.m. is shown, n = 9 cells from 6 separate experiments for both Wt and PAK1−/− BMMs.

Fig. 4

Fig. 4

PAK1 promotes ERK1/2 activation at the cell periphery. A, Wt and PAK1−/− BMMs were adhered onto tissue culture plastic for 10 minutes in growth medium. Lysates were immunoblotted for phospho-Thr202/Tyr204-ERK1/2, phospho-Thr180/Tyr182-p38, phospho-Ser298-MEK1/2 and total ERK1/2. Densitometry quantification of phosphorylated ERK1/2 and p38 levels equalised to total ERK1/2 protein levels are shown (a.u., arbitrary units). Data show the mean +/− s.d. of two separate experiments. B, Wt and PAK1−/− BMMs were plated onto glass coverslips for 10 minutes in growth medium and were stained using an ERK1/2 antibody and TRITC-phalloidin to visualise F-actin. Cells were imaged by confocal microscopy. ERK1/2 localisation was quantified by determining the number of cells with ERK1/2 staining at the periphery. The mean +/− s.d. is shown for two separate experiments, n = 60 (Wt) and 45 (PAK1−/−). C, BMMs were stained with a phospho-Thr202/Tyr204-ERK1/2 antibody (P-ERK1/2) and TRITC-phalloidin (F-actin). Localisation of phospho-ERK1/2 was quantified by determining the number of cells with staining at the cell periphery. The mean +/− s.d. is shown for two separate experiments, n = 39 (Wt) and 62 (PAK1−/−). D, Wt and PAK1−/− BMMs were kept in suspension or adhered onto tissue culture plastic for 10 minutes in growth medium. Lysates were immunoblotted for phospho-Ser217/221-MEK1/2, and total ERK1/2 as a loading control. Densitometry quantification of phosphorylated MEK1/2 equalised to total ERK1/2 protein levels is shown (a.u., arbitrary units). Data show the mean +/− s.d. of two separate experiments.

Fig. 5

Fig. 5

Inhibition of ERK1/2 activation promotes macrophage spreading. Wt BMMs were plated on glass coverslips in the presence or absence of 1 μg/ml U0126. A, Representative images of cells stained for F-actin. B, Cell spread area was quantified at the indicated time-points. The mean spread area +/− s.e.m. from three separate experiments is shown. ***p<0.001 comparing Wt + U0126 to untreated Wt, n ~ 50 cells/timepoint/experiment.

Fig. 6

Fig. 6

Inhibition of ERK1/2 activation reduces lamellipodial stability. A, Wt BMMs in growth medium were pre-treated with 1 μg/ml U0126 for 1 hour and plated on to glass-bottomed tissue culture dishes. Time-lapse microscopy was used to monitor cell spreading. Frames from the indicated time-points are shown. Bar = 20 μm. B, Quantification of the number of lamellipodia per cell observed in the time-lapse movie frames specified. Data are the mean +/− s.e.m. from three separate experiments. C, Representative kymograph of a lamellipodium in a U0126-treated cell. The inset shows the kymograph with the membrane edge highlighted (white line).

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