PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells

Abstract

p21-Activated kinase 4 (PAK4), a serine/threonine kinase, is purported to localize to podosomes: transient adhesive structures that degrade the extracellular matrix to facilitate rapid myeloid cell migration. We find that treatment of transforming growth factor β (TGF-β)-differentiated monocytic (THP-1) cells with a PAK4-targeted inhibitor significantly reduces podosome formation and induces the formation of focal adhesions. This switch in adhesions confers a diminution of matrix degradation and reduced cell migration. Furthermore, reduced PAK4 expression causes a significant reduction in podosome number that cannot be rescued by kinase-dead PAK4, supporting a kinase-dependent role. Concomitant with PAK4 depletion, phosphorylation of Akt is perturbed, whereas a specific phospho-Akt signal is detected within the podosomes. Using superresolution analysis, we find that PAK4 specifically localizes in the podosome ring, nearer to the actin core than other ring proteins. We propose PAK4 kinase activity intersects with the Akt pathway at the podosome ring:core interface to drive regulation of macrophage podosome turnover.

Keywords: AKT; PAK4; adhesion; migration; podosome; superresolution microscopy.

Graphical abstract

Figure 1

PAK4 Kinase Activity Drives Podosome Formation (A) Confocal images of THP-1 cells seeded on fibronectin with TGF-β for 16 h and then treated for 4 h with DMSO control or 1 μM PAK inhibitors PAK4i or IPA-3. Stained for vinculin (green) and F-actin (red). Insert zoom of podosomes or peripheral adhesions. (B and C) Percentage of THP-1 cells with podosomes following treatment with DMSO or 1 or 5 μM PAK inhibitors for 4 h (B) and the percentage of cells with 0, 1–10, 11–20, 21–30, or ≥31 podosomes per cell was calculated from 300 cells per condition (C). (D) Following a 4 h treatment with DMSO or inhibitors, the inhibitors were washed out and cells incubated with TGF-β for a further 4 h. Cells were fixed and stained for vinculin and F-actin at times indicated, and the percentage of cells with podosomes were counted. (E) Primary human monocytes isolated from peripheral human blood from healthy donors were seeded on fibronectin and differentiated toward macrophages by incubating for 4.5 days with 50 ng/ml M-CSF. Macrophages were then treated with DMSO or 1 μM PAK inhibitor PAK4i or IPA-3, fixed, and stained for vinculin (green) and F-actin (red). (F) Percentage of primary human macrophages with podosomes following a 4 h treatment with DMSO or 1 or 5μM PAK inhibitors. (G) Western blot for PAK1 and PAK4 levels in lysates of primary monocytes from two healthy donors (HD1 and HD2) cultured for 6 days, alongside a THP-1 lysate from cells differentiated for 16 h with TGF-β. For all graphs, error bars represent ±SEM, and p values indicate significant difference to DMSO-treated cells by one-way ANOVA. Scale bars in (A) and (E) represent 10 μm.

Figure 2

Podosome Loss Following PAK Inhibition Is Accompanied by an Increase in Focal Adhesions and a Reduction of Invasive Migration (A) For the matrix degradation assay, >20 fields of view per treatment condition were measured. (B) Mean cell speed (μm/minute) was calculated from >90 cells from three separate experiments. (C) Confocal images of THP-1 cells seeded on fibronectin with TGF-β for 16 h and then treated for 4 h with 1 μM PAK inhibitors. Cells were stained for zyxin (green), vinculin (red), and F-actin (blue). Insert zoom of peripheral adhesion. Scale bars represent 10 μm. (D) Percentage of cells with focal adhesions. (E) Number of focal adhesions/cell. For all graphs, error bars represent ±SEM, and p values denote significant difference to DMSO-treated cells by one-way ANOVA.

Figure 3

PAK4 Knockdown and Rescue Supports a Kinase-Dependent Role for PAK4 in Podosomes through the Activation of Akt (A) PAK4 shRNAs or scrambled control cells were probed for PAK4 expression. (B) PAK4 shRNA-expressing cells were seeded on fibronectin with TGF-β for 16 h, fixed, and stained for vinculin (green) and F-actin (red). (C) Percentage of cells with podosomes (>300 cells per cell line). (D) PAK4 knockdown cells (A) expressing EGFP-tagged shRNA-resistant PAK4: EGFP-PAK4 (rescue) or the kinase dead mutant PAK4: EGFP-PAK4r (K350,351M) were probed for PAK4 expression. (E) Confocal images of PAK4 rescue THP-1 cells. (F) Percentage of cells with podosomes. (G) Cells were probed for pAkt and PAK4. (H) THP-1 cells on fibronectin with TGF-β for 16 h were treated with indicated concentrations of Akt inhibitor for 4 h, fixed and stained for F-actin, and scored for podosomes. (I) Confocal images of THP-1 cells fixed and stained for pAkt, F-actin, and vinculin. Error bars = ± SEM and p values indicate significant differences between treated cells by one-way ANOVA. Scale bars in (B) and (E) = 10 μm; scale bars in (I) = 5 μm.

Figure 4

PAK4 Localizes to the Podosome Ring (A and B) THP-1 cells stably expressing EGFP or EGFP-PAK4 were seeded on fibronectin with TGF-β for 16 h prior to immunoprecipitation (IP) of paxillin (A) or vinculin (B). Blots were probed for endogenous PAK4 and reprobed for GFP and paxillin or vinculin. (C) EGFP-PAK4-expressing THP-1 cells were seeded on fibronectin with TGF-β for 16 h, fixed, and stained for vinculin. Datasets of 200 images were taken for both EGFP-PAK4 and vinculin and analyzed using the ImageJ 3B plugin. Left panels are reconstructed localizations from two adjacent podosomes (reconstruction blur FWHM = 20 nm), and these localizations are representative of >50 podosomes analyzed using 3B; scale bar represents 500 nm. Right panel shows a reconstructed dataset from 3B analysis carried out using a computer cluster to give localizations in podosomes of an entire THP-1 cell; scale bar represents 2 μm. (D) STORM and 3B localization of F-actin and EGFP-PAK4, respectively. Scale bar represents 5 μm. (E–G) 3B datasets generated for >50 podosomes from >10 EGFP-PAK4-expressing THP-1 cells stained for vinculin (E), paxillin (F), or co-expressing mCherry-Talin (G) were analyzed using ring analysis software (Staszowska et al., 2017). Histograms show the absolute distances from the podosome center of EGFP-PAK4 (blue) and vinculin/paxillin/mCherry-Talin (red). (H) A top-down representation of the relative localizations of PAK4 and the podosome ring proteins vinculin, paxillin, and talin, based on 3B localizations.