Signalling by the non-conventional p21-activated kinase 4

Ph.DDOCTOR OF PHILOSOPH

PAK4 regulates centrosomal reorientation in U2OS cells.

<p>A) Diagram showing how cells were scored for forward centrosomal reorientation based on immuno-localization of γ-tubulin (prominent red dot) and the nucleus (blue). This was done using a 120° sector (indicated in yellow) centered on the nucleus, and cells with γ-tubulin placed within the forward sector facing the wound edge are scored (as marked with asterisks). The wound is toward the top of the figure and the image was acquired 1h post wounding. B) Graph showing the percentage of U2OS cells with forward centrosome orientation 1h post wounding. Cells were transfected with siRNA to Cdc42, Rac1 or PAK4 and left for 48h before analysis. Cells were also treated with either a PAK4 inhibitor PF-3758309 or a PKC inhibitor GF109203X for 1h prior to scratching. The data represents three independent experiments (N = 60) with standard error of mean. A random orientation of the centrosome gives a 33% baseline (solid black line). C) Graph showing the same analysis performed with COS-7 cells 1h post wounding. Cells were transfected 48h with siPAK4 or treated with inhibitors to PAK4 (PF-3758309) or PKC (GF109203X) for 1h before scratching. *<i>P</i> value < 0.01, **<i>P</i> value < 0.005. Scale bar: 10μm.</p

PAK4 phosphorylates β-catenin at Ser-675.

<p>A) U2OS or LNCaP cells were transfected overnight with FLAG-PAK4 S445N (denoted PAK4*), or with PAK4 siRNA or Cdc42 siRNA for 48h. The cell lysates (30 μg per lane) processed for Western blotting are as indicated. B) The signal intensities corresponding to bands detected by anti-pSer-675 β-catenin or total anti-β-catenin were obtained from 3 independent experiments using ImageJ, averaged and plotted with standard error of mean. *<i>P</i> value < 0.05. C) U2OS cells were transfected with GFP or GFP-Cdc42(G12V) (GFP-Cdc42*), and fixed the following day. Cells were then immuno-stained for PAK4 and the nuclei were stained by DAPI. Two neighbouring cells in which the cell-cell junction was observed are denoted by asterisks. Scale bar: 10μm.</p

Inhibition of PAK4 affects β-catenin Ser-675 phosphorylation.

<p>A) U2OS cells were treated with an inhibitor to PAK4 (5 μM PF-3758309) or vehicle for 1h before fixation, and then immuno-stained for PAK4 and p120-catenin. B) U2OS cells were similarly treated as in (A), but immuno-stained for pSer-675 β-catenin and p120-catenin instead. C) U2OS cells were treated with an inhibitor to PKA (10 μM H-89) or 5 μM PF-3758309 for 1h before lysates were harvested and subjected to Western blotting. The PAK4 bands are marked by arrowheads. D) Band signal intensities of β-catenin pSer-675 against total β-catenin was obtained from Western blots in 3 independent experiments via analysis using ImageJ, averaged and plotted with standard error of mean. *<i>P</i> value < 0.05. Scale bar: 10μm.</p

Endogenous PAK4 localization to cell-cell junctions is dependent on Cdc42.

<p>A) Cells from different cell lines were plated to confluency onto coverslips and fixed. Fixed cells were immuno-stained with PAK4 and adherens junction marker p120-catenin antibodies. B) Processed cells were imaged under high resolution confocal microscopy to visualize the localization of PAK4 with regard to p120-catenin. C) U2OS or COS-7 cells were transfected with PAK4 or Cdc42 siRNA for 48h and probed for levels of PAK4 and Cdc42 by Western analysis on Triton X-100 soluble cell lysates (30 μg per lane). D) U2OS cells were transfected with Cdc42 siRNA or PAK4 siRNA, fixed after 48h and then immuno-stained for the presence of PAK4 and p120-catenin. Scale bar: 10 μm.</p

The Cdc42 Effector Kinase PAK4 Localizes to Cell-Cell Junctions and Contributes to Establishing Cell Polarity

<div><p>The serine/threonine kinase PAK4 is a Cdc42 effector whose role is not well understood; overexpression of PAK4 has been associated with some cancers, and there are reports that correlate kinase level with increased cell migration <i>in vitro</i>. Here we report that PAK4 is primarily associated with cell-cell junctions in all the cell lines we tested, and fails to accumulate at focal adhesions or at the leading edge of migrating cells. In U2OS osteosarcoma and MCF-7 breast cancer cell lines, PAK4 depletion did not affect collective cell migration, but affected cell polarization. By contrast, Cdc42 depletion (as reported by many studies) caused a strong defect in junctional assembly in multiple cells lines. We also report that the depletion of PAK4 protein or treatment of cells with the PAK4 inhibitor PF-3758309 can lead to defects in centrosome reorientation (polarization) after cell monolayer wounding. These experiments are consistent with PAK4 forming part of a conserved cell-cell junctional polarity Cdc42 complex. We also confirm β-catenin as a target for PAK4 in these cells. Treatment of cells with PF-3758309 caused inhibition of β-catenin Ser-675 phosphorylation, which is located predominantly at cell-cell junctions.</p></div

Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea

International audienceAsgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures

PAK4 does not localize to the leading edge of migrating cells.

<p>A) Total lysates (30 μg per lane) extracted from HeLa, COS-7 and U2OS cells were probed for PAK4 by Western blotting using either an affinity purfied PAK4 antibody [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129634#pone.0129634.ref014" target="_blank">14</a>] or Cell Signaling (*3242). Recombinant purified His-tagged PAK4 (6XHis-PAK4) in lane 1 was loaded (10 ng) as a size control. B) Images showing the typical morphology of migrating U2OS cells at the wound-edge in a standard monolayer scratch assay. The cells were fixed with methanol and immuno-stained with anti-PAK4 and anti-p120-catenin. PAK4 enrichment at the cell-cell junction is indicated by white arrows (top panel), and the edge of tone of the lamellipodia is marked with a white dotted line (bottom panel). Scale bar: 5 μm.</p

PAK4 loss does not affect collective migration rates of U2OS or MCF-7 cells.

<p>A) (Left panel) U2OS or MCF-7 cells were transfected with siRNA directed to PAK4 or Cdc42 as indicated. The cell lysates (30 μg per lane) were probed for expression of PAK4, Cdc42 or tubulin. (Right panel) U2OS cells were transfected with siRNA directed to Rac1 as indicated and the lysates were probed for expression of Rac1 or tubulin. B) Low power images of the same area of the monolayer scratch wound are shown before and after 4h cell migration into the gap. The wound-edge is represented in yellow and red corresponding to the start and end of imaging respectively. C) Bar chart depicting the area covered over 4h after the scratch was applied by either the U2OS or MCF-7 cells, with standard error of mean. The area was calculated using ImageJ software. *<i>P</i> value < 0.05, **<i>P</i> value < 0.005. Scale bar: 50μm.</p

Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea

Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures