PMC

Fig. 5. Activated Tek promotes the formation of a Pak, Nck and Dok-R ternary complex in vivo and results in Pak activation. (A) SVRs were stimulated with Ang1-MH or DMEM in the presence of sodium orthovanadate. Immunoprecipitation of Dok-R resulted in co-immunoprecipitation of Pak in lysates from Ang1-MH-stimulated cells. Reprobing the membrane with anti-Dok-R antibodies revealed equal amounts of immunoprecipitated Dok-R from both stimulated and unstimulated lysates. (B) Lysates from HEK 293T cells overexpressing Pak, Tek/Tek^K853A and Dok-R/Dok-R^Y351F were immunoprecipitated with antibodies that specifically recognize Dok-R, Pak or Tek. In cells overexpressing Tek + Dok-R, both Dok-R and Pak could co-immunoprecipitate with one another. Interaction of Dok-R with Pak was not observed in cells expressing either a kinase inactive Tek (Tek^K853A) or Dok-R^Y351F. Immunoblotting of cell lysates (CL) revealed equal expression of Pak, Dok-R and Tek. (C) Pak immunoprecipitates from the above transfectants were subjected to an in vitro kinase assay using MBP as substrate. Tek-mediated MBP phosphorylation by Pak was 40% greater from cells that expressed Dok-R than from cells expressing Dok-R^Y351F or Tek^K853A with either Dok-R or Dok-R^Y351F. The graph represents MBP phosphorylation values after normalization for Pak immunoprecipitation. Immunoprecipitation of Tek and Tek^K853A with anti-Tek antibodies revealed that Tek, but not Tek^K853A, was tyrosine phosphorylated. Data shown are average of two independent experiments ± SD.

Stimulatory and inhibitory effects of P. aeruginosa on NF-κB in Calu-3 cells. PAK, PAK ΔfliC, and heat-killed (80°C, 1 h) PAK ΔfliC at the doses shown were added to NF-κB-luc-expressing Calu-3 cells grown in medium for 4 h in a 37°C incubator. (A) Effects of PAK, PAK ΔfliC, and heat-killed PAK ΔfliC. Luciferase activity was expressed relative to the maximal activity attained during treatment with PAK, which averaged a 6.2-fold ± 0.4-fold increase compared to that of untreated control Calu-3 cells. The symbols * and # indicate significant difference from the value measured for untreated control cells. n = 3 to 6 for all data. (B) Effects of PAK, PAK ΔfliC, and heat-killed PAK ΔfliC on NF-κB in flagellin-stimulated Calu-3 cells. Luciferase activity was expressed relative to the maximal activity attained during treatment with flagellin (10⁻⁷ g/ml) or flagellin plus PAK. The response to flagellin averaged a 6.1-fold ± 0.6-fold increase compared to that of untreated control Calu-3 cells. In the presence of flagellin, PAK inhibited NF-κB compared to flagellin alone, and PAK ΔfliC caused even larger inhibitions of flagellin-stimulated NF-κB activity. Heat-killed PAK ΔfliC (open square) did not inhibit the response to flagellin. Data are averages ± standard deviations (n = 3). The symbol * indicates significant difference (P < 0.05) from flagellin-treated cells. (C) Inhibitory effect of PAK ΔfliC on flagellin-stimulated IL-8 secretion. Calu-3 cells or primary human tracheal epithelial cells (hTE) were left untreated or were treated with flagellin (10⁻⁷ g/ml), PAK ΔfliC (10⁸ CFU/ml), or flagellin plus PAK ΔfliC for 4 h. Bathing media were sampled at t = 0 and after 4 h, and IL-8 was measured. PAK ΔfliC had no effect on IL-8 secretion by control cells but reduced the response to flagellin. Results are averages ± standard deviations (n = 3). The symbol * indicates significant difference from flagellin-treated cells.

A to H: Level of S100A7 tissue production (red) assessed by immunohistochemistry on RHE infected with the different PAK strains of P. aeruginosa for 24 h. Nuclei were counter-stained using a hematoxylin solution (blue). A: control (sterile PBS), B: wild-type PAK strain, C: PAK ∆fliC (Fla⁻), D: PAK ∆xcpQ (T2SS⁻), E: PAK ∆pscF (T3SS⁻), F: PAK ∆fliC∆xcpQ∆pscF (Fla⁻ /T2SS⁻/T3SS⁻), G: ultrapure flagellin deposited in culture medium, H: ultra-pure flagellin deposited on the upper layer of RHE. I to P: Immunofluorescence assays (green) using a polyclonal anti-P. aeruginosa antibody on RHE infected with the different PAK strains. Nuclei were counter-stained using a solution of propidium iodide red-fluorescent staining. I: control (sterile PBS), J: wild-type PAK strain, K: PAK ∆fliC (Fla⁻), L: PAK ∆xcpQ (T2SS⁻), M: PAK ∆pscF (T3SS⁻), N: PAK ∆fliC∆xcpQ∆pscF (Fla⁻ /T2SS⁻/T3SS⁻), O: ultrapure flagellin deposited in culture medium, P: ultra-pure flagellin deposited on the upper layer of RHE.

(A) Representative confocal images of ventral muscle fields in late embryos (21 hours after egg laying) and early first instar larvae (2 hours after hatching) of indicated genotypes labeled for Brp (red), PAK (green), and HRP (blue). The synaptic PAK signals are weak at neto-β mutant NMJs, but normal at the muscle attachment sites. (B) Confocal images of NMJ4 in third instar larvae of indicated genotypes labeled for PAK (red), GluRIIA (green) and HRP (blue). Muscle expression of a constitutively membrane bound form of PAK (G14> pak ^myr) does not rescue the GluRIIA synaptic abundance at neto ^βshort NMJs compared to control (G14 /+). PAK signals remain diffuse and localize perisynaptically in the absence of an intact Neto-β (see bouton details). (C) Confocal images of NMJ4 in control (w ¹¹¹⁸) and various pak heteroallelic third instar larvae labeled for Neto-ex (red), PAK (green) and HRP (blue). Lack of PAK does not impact the synaptic distribution of Neto-β. Scale bars: (A) 10 μm; (B-C) 20 μm, 2 μm in details.