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doi: 10.1242/10.1242/jcs.00080

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PAK4 is activated via PI3K in HGF-stimulated epithelial cells

Claire M. Wells¹, Arie Abo^2,* and Anne J. Ridley^1,3,

¹ Ludwig Institute for Cancer Research, Royal Free and University College Medical School Branch, 91 Riding House Street, London WIW 7BS, UK
² Onyx Pharmaceuticals, 3031 Research Drive, Richmond, CA 94806, USA
³ Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, UK
^* Present address: PPD Discovery, 1505 O'Brien Dr Menlo Park, CA 94025, USA

View larger version (24K): [in a new window]   Fig. 1. PAK4 expression and activity in MDCK cells. (A) Analysis of endogenous PAK isoform expression in MDCK cell lysates. A western blot of MDCK cell lysates was probed with antibodies against PAK4 (lane 1) and PAK1, -2 and -3 (lane 2). (B) Kinase activity of PAK4. MDCK cells were transfected with HA-PAK4wt (Wt), HA-PAK4 () and HA-PAK4GBD (GBD) and incubated for 24 hours in 0.2% FCS. PAK4 was immunoprecipitated from cell lysates with anti-HA mAb and its kinase activity assayed in the presence of Histone H1 and [-32P]ATP. Substrate phosphorylation was analysed after SDS-PAGE and autoradiography. Phosphorylation of Histone (arrow) and autophosphorylation (arrowhead) is indicated. Retained cell lysates were subjected to western blotting with the anti-HA mAb to show expression levels of exogenous PAK4 proteins. Autoradiographs and western blots were quantified using Kinetic Imaging software and the level of kinase activity normalised to protein expression levels. The results shown are the means±s.e.m. of three independent experiments.

View larger version (61K): [in a new window]   Fig. 2. Activated PAK4 induces cell rounding. (i) MDCK cells were microinjected with a plasmid encoding PAK4GBD and incubated for 3 hours in 0.2% FCS prior to a further incubation in 0.2% FCS for 20 hours (A) or stimulation with HGF for 20 hours (B). Cells were then fixed and stained for HA-tagged PAK4 (green) and F-actin (red). Bar, 10 µm. Apical sections (at the level of F-actin-containing microvilli) are shown of PAK4GBD expression (B) in order to visualise clearly any rounded-up PAK4-expressing cells (arrows). In this plane non-expressing rounded cells are primarily mitotic cells. (ii) MDCK cells were microinjected with a plasmid encoding the kinase domain of PAK4, PAK4 (A,C), or a kinase-inactive PAK4 kinase domain, PAK4M350 (B), and incubated for 3 hours in 0.2% FCS prior to stimulation for 20 hours with HGF (A,B) or further incubation in the absence of HGF (C). Cells were fixed and stained for HA-tagged PAK4 (A-C) and F-actin (D-F). Bar, 10 µm. (iii) Selected frames (time=0 or 4 hours) are shown from a time-lapse video recording (Movie 1) of MDCK cells expressing activated PAK4 (PAKGBD) (arrows) and stimulated at t=0 with HGF. Bar, 20 µm.

View larger version (90K): [in a new window]   Fig. 3. PAK4 induces loss of paxillin-associated focal complexes. MDCK cells were microinjected with a plasmid encoding either PAKGBD or PAK4wt and incubated for 3 hours in 0.2% FCS before stimulation for 3 hours with HGF. Cells were then fixed and stained for HA-tagged PAK4 (A,B) and paxillin (C,D). Arrows indicate the absence of paxillin-associated focal complexes in a PAKGBD-expressing cell (C) and the presence of paxillin-associated focal complexes in a cell expressing PAK4wt (D). Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 4. PAK4 is localised at the cell periphery. MDCK cells were microinjected with plasmids encoding PAK4GBD or PAK4wt and incubated for 5.5 hours in 0.2% FCS (A,C) or 3 hours in 0.2% FCS prior to stimulation with HGF for 2.5 hours (B,D). (E) MDCK cells were microinjected with a plasmid encoding PAK4 and incubated for 4.5 hours in 0.2% FCS. Cells were then fixed and stained for HA-tagged PAK4 and F-actin. Bar, 10 µm. The arrow (F) indicates a PAK4GBD-expressing cell; arrowheads (D) indicate PAK4wt localisation at the periphery.

View larger version (32K): [in a new window]   Fig. 5. PAK4 kinase activity is elevated by HGF. (A) MDCK cells were transfected with HA-PAK4wt and serum-starved for 4 hours prior to stimulation with HGF for the indicated times. PAK4 was immunoprecipitated from cell lysates with anti-HA mAb and its kinase activity assayed in the presence of Histone H1 and [-32P]ATP. Phosphorylation of Histone is indicated. As a control (C) in each experiment (A-D), a kinase assay was also performed on protein G-Sepharose beads coupled to anti-HA mAb, which had been incubated with untransfected cell lysate. Autoradiographs and western blots were quantified using Kinetic Imaging Software and the level of kinase activity normalised to protein expression levels. (B,C) MDCK cells were transfected with HA-PAK4GBD or HA-PAK4wt and serum-starved for 4 hours prior to stimulation with HGF for 15 minutes. LY294002 was added to cultures after 3.5 hours of serum starvation. PAK4 was immunoprecipitated from cell lysates with anti-HA mAb and its kinase activity assayed in the presence of Histone H1 and [-32P]ATP. Autoradiographs and western blots were quantified as above. Time in the presence of HGF in minutes is indicated; LY, LY294002. The results shown are mean±s.e.m. of three independent experiments. (D) MDCK cells were transfected with HA-PAK4wt and HA-PAK4M350 and starved for 4 hours. PAK4 proteins were immunoprecipitated from cell lysates with anti-HA mAb and their kinase activity assayed in the presence of Histone H1 and [-32P]ATP.

View larger version (78K): [in a new window]   Fig. 6. PAK4-induced rounding and PAK4wt localisation is prevented by PI 3K inhibitors (A) Cells were microinjected with a plasmid encoding PAK4GBD and incubated for 2.5 hours in 0.2% FCS. Cells were then incubated for 30 minutes with LY294002. Cells were then incubated for 20 hours in the presence of HGF and LY294002. (B) Cells were microinjected with a plasmid encoding PAK4 and incubated for 2.5 hours in 0.2% FCS. Cells were then incubated for 30 minutes with LY294002, prior to further incubation in 0.2% FCS plus LY294002 for 20 hours. (C) Cells were microinjected with a plasmid encoding PAK4GBD and incubated for 2 hours in 0.2% FCS. Cells were then incubated for 30 minutes with LY294002 prior to a further incubation in 0.2% FCS plus LY294002 for 2.5 hours. (D) Cells were microinjected with a plasmid encoding PAK4wt and incubated for 2 hours in 0.2% FCS. Cells were then incubated for 30 minutes with LY294002 prior to stimulation for 2.5 hours with HGF and LY294002. All the cells were fixed and stained for HA-tagged PAK4 and F-actin (E-H). Basal sections (at the level of stress fibres) of PAK4GBD-expressing cells are shown, while apical sections (at the level of F-actin-containing microvilli) of PAK4-expressing cells are shown in order to visualise clearly the rounded-up cells (arrows). Bar, 10 µm.