Novel p47(phox)-related organizers regulate localized NADPH oxidase 1 (Nox1) activity

Abstract

The mechanisms that determine localized formation of reactive oxygen species (ROS) through NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox) family members in nonphagocytic cells are unknown. We show that the c-Src substrate proteins Tks4 (tyrosine kinase substrate with four SH3 domains) and Tks5 are functional members of a p47(phox)-related organizer superfamily. Tks proteins selectively support Nox1 and Nox3 (and not Nox2 and Nox4) activity in reconstituted cellular systems and interact with the NoxA1 activator protein through an Src homology 3 domain-mediated interaction. Endogenous Tks4 is required for Rac guanosine triphosphatase- and Nox1-dependent ROS production by DLD1 colon cancer cells. Our results are consistent with the Tks-mediated recruitment of Nox1 to invadopodia that form in DLD1 cells in a Tks- and Nox-dependent fashion. We propose that Tks organizers represent previously unrecognized members of an organizer superfamily that link Nox to localized ROS formation.

Figure 1. Tks4 and Tks5 are novel members of the p47 organizer superfamily and support Nox1-dependent ROS generation in a reconstituted HEK293 cell system

(A). Schematic diagram of the members of the p47 organizer superfamily. The white squares indicate PX domains, while white circles indicate SH3 domains. (B). Time course of ROS generation examining the ability of Tks4 and Tks5 to support Nox1-dependent ROS formation in a reconstituted HEK293 system. HEK293 cells were transfected as indicated with expression vectors for Nox1, NoxA1 and RacQL, and with different organizers, including NoxO1, Tks4, Tks5 or empty vector. After 24hrs, ROS production was measured by a luminol-based chemiluminescence (CL)-assay continuously for 30 minutes. One representative experiment from three separate experiments is shown. All transfected proteins were expressed to similar levels, as per Fig. S1.

Figure 2. Tks4 and Tks5 selectively support Nox1- and Nox3-dependent ROS generation in a Rac GTPase-dependent manner

(A). CL-assay to compare the ability of Tks4 and Tks5 to support ROS generation by different members of the NADPH oxidase family in an HEK293 cell reconstituted system. HEK293 cells were transfected with expression vectors for the indicated organizer subunits and with the known components required for Nox1- (=Nox1, NoxA1, RacQL), Nox2- (=Nox2, p67^phox, RacQL), Nox3- (=Nox3, NoxA1, RacQL) and Nox4- (=Nox4)-dependent ROS generation. After 24hrs, ROS formation was monitored by CL-assay. One representative experiment from three separate experiments is shown, and results are given as mean of triplicates +/− S.D. (B) Treatment with the flavoenzyme/Nox inhibitor diphenyliodonium (DPI) blocks Tks4- and Tks5-mediated ROS generation by Nox1 in the HEK293 reconstituted system. HEK293 cells were transfected with expression vectors for different Myc-tagged organizer subunits as indicated, and with all of the components required for Nox1 activity. 24 hrs after, cells were treated with 10μM DPI or DMSO control for 30 minutes and ROS production was measured by CL-assay (upper panel). Comparable expression of Myc-tagged proteins was verified by Western blot (lower panel). One representative experiment from three separate experiments is shown, and results of CL-assay are given as mean of triplicates +/− S.D. (C) Tks4- and Tks5-mediated ROS formation by Nox1 is Rac-dependent in the reconstituted HEK293 cell system, as demonstrated using dominant negative Rac1T17N, the non-Rac-binding R103E NoxA1 mutant, and the non-Rac-binding Nox1 K421A, Y425A, K426E triple mutant (Nox1TM). HEK293 cells were transfected as indicated and after 24hrs ROS production was monitored by CL-assay (upper panels). The expression of Myc-tagged proteins was verified by Western blot (lower panels). One representative experiment from three separate experiments is shown, and results of CL-assays are given as mean of triplicates +/− S.D.

Figure 3. Support of Nox1-dependent ROS generation by Tks4 and Tks5 requires the SH3 domain-mediated binding of NoxA1 and is independent of Rac

(A) Co-immunoprecipitation analysis indicates that Tks4 and Tks5 interact with NoxA1 in reconstituted HEK293 cells and with endogenous NoxA1 in DLD1 cells. In the left panel, HEK293 cells were transfected as indicated with Myc-tagged organizer subunits and Flag-tagged NoxA1. After 24hrs, cells were lysed and immunoprecipitation was carried out (see Methods) using anti-Myc antibody. Interaction of Myc-tagged adaptors with NoxA1 was analyzed using anti-NoxA1-specific antibody, while expression of transfected proteins in cell lysates and equal loading of proteins on gels was verified by re-blotting the membranes with anti Myc- and actin-antibody, respectively. One representative experiment from three separate experiments is shown. In the right panel, DLD1 cells were lysed in RIPA buffer and immunoprecipitation was performed using NoxA1-specific antibody or pre-immune serum. An additional control with protein G-Sepharose beads only was also carried out. Specific interaction between endogenous Tks4 and NoxA1 was confirmed using Tks4-specific antibody, while the presence of NoxA1 in cell lysates was verified by re-blotting the membrane with NoxA1-specific antibody. One representative experiment from three separate experiments is shown. (B) Co-immunoprecipitation analysis indicates that the interaction between NoxA1 and Tks5 is not dependent of the GTP-bound state of Rac1. HEK293 cells were transfected as indicated with Myc-tagged Tks5, Flag-tagged NoxA1, and alternatively with GFP-tagged Rac1-Q61L or Rac1-T17N. After 24hrs, cells were lysed and immunoprecipitation was carried out using anti-Flag antibody. Interaction of Tks5 with NoxA1 was analyzed using anti-Tks5-specific antibody, while expression of transfected proteins in cell lysates and similar amount of immunoprecipitated NoxA1 protein was verified by re-blotting the membranes with anti NoxA1- and GFP-antibody. One representative experiment from three separate experiments is shown (C) Tks5 binds to NoxA1 through its SH3 domains. HEK293 cells were transfected with expression vectors for Nox1, NoxA1, RacQL and with the Myc-tagged organizer subunits NoxO1, Tks5 wild-type, or Tks5 M1M5 mutant in which point mutations were made to inactivate all of the SH3 domains. After 24hrs, ROS generation was monitored by CL-assay (left panel) and expression of transfected proteins was checked by Western blot using Tks5-specific antibody (right panel). The loss of the ability of the Tks5 M1M5 mutant to bind NoxA1 was shown by co-immunoprecipitation analysis using Flag antibody for immunoprecipitation and Tks5 antibody to detect specific interaction (lower panel). One representative experiment from three separate experiments is shown, and results of CL-assays are given as mean of triplicates +/− S.D. (D) The deletion of the PX domain of Tks5 prevents the activation of Nox1-mediated ROS formation, but does not abrogate the ability of Tks5 to bind NoxA1. HEK293 cells were transfected with expression vectors for the Myc-tagged organizer subunits NoxO1, Tks5 wild-type, and Tks5ΔPX, along with Flag-tagged NoxA1. After 24hrs, ROS generation was measured by CL-assay (left panel), while expression of Myc-tagged organizers was verified by Western blot using Myc antibody (right panel). The ability of Tks5ΔPX to bind NoxA1 was determined by co-immunoprecipitation analysis (lower panel) using Flag antibody for immunoprecipitation and Tks5 antibody to detect interaction with NoxA1. One representative experiment from three separate experiments is shown, and results of CL-assays are given as mean of triplicates +/− S.D.

Figure 4. Human DLD1 colon cancer cells produce ROS and degrade the ECM in a Tks4-dependent manner

(A) Tks4 knockdown by siRNA reduces ROS generation in a concentration-dependent fashion and this effect can be rescued by a siRNA-insensitive mouse Tks4. In the upper panels, DLD1 cells were transfected with different concentrations of Tks4-specific siRNA mixture or control-siRNA and after 72hrs, ROS generation was determined by CL-assay (left panel), while the extent of Tks4 protein knockdown was checked by Western blot using Tks4-specific antibody (right panel). In the lower panels, DLD1 cells were transfected with 20nM of Tks4-specific siRNA mixture or control siRNA and with mouse Tks4 (mTks4) expression vector or empty vector. After 72hrs, ROS generation was measured by CL-assay (left panel) while Tks4 knockdown and mTks4 expression were checked by Western blot using Tks4-specific antibody (right panel). One representative experiment from three separate experiments is shown and results of CL-assays are given as mean of triplicates +/− S.D. (B) Tks4 knockdown by siRNA reduces Rac1-induced ROS generation and this effect can be rescued by overexpression of mTks4. DLD1 cells were transfected with Tks4-specific siRNA or control siRNA and with expression vector for GFP-tagged RacQL or empty vector. After 72hrs, ROS generation was monitored by CL-assay (left panel). Tks4 protein knockdown and mTks4 expression, as well as comparable expression levels of GFP-tagged RacQL were demonstrated by Western blot using Tks4- or GFP-specific antibody, respectively (right panel). One representative experiment from three separate experiments is shown and results of CL-assays are given as mean of triplicates +/− S.D. (C) Tks4 and Tks5 overexpression in DLD1 cells induces ROS generation to a similar extent as NoxO1 overexpression (2-fold over control). DLD1 cells were transfected with expression vectors for Myc-tagged organizer subunits (NoxO1, Tks4 or Tks5) or with empty vector. After 24hrs, ROS formation was measured by CL-assay (left panel), while similar expression levels of the Myc-tagged adaptors were verified by Western Blot using anti-Myc antibody (right panel). One representative experiment from three separate experiments is shown and results of CL-assays are given as mean of triplicates +/− S.D. (D) Tks4 knockdown reduces the ability of DLD1 cells to degrade the extracellular matrix (ECM). DLD1 cells were transfected with Tks4-specific or control siRNA and after 24hrs plated on FITC-labeled gelatin-coated coverslips. After 20hrs, cells were fixed in 4% PFA, stained with Alexa-Fluor-568 phalloidin as described in Methods, and visualized by epifluorescence microscopy (40X). White arrows indicate areas in which F actin-positive structures (in red) degrade the ECM. Scale bars, 45μm. One representative picture from two separate experiments is shown.

Figure 5. Nox1 localizes to ECM-degrading invadopodia in DLD1 cells

(A) and (B) Nox1 localizes to F actin- and cortactin-rich structures in DLD1 cells. DLD1 cells were plated on glass coverslips and after 24hrs cells were transfected with active SrcYF and GFP-tagged Nox1 or GFP empty vector. After 48hrs the cells were fixed in PFA4% and stained with Alexa-Fluor-568 phalloidin (A) or cortactin antibody, followed by Alexa-Fluor 568-conjugated secondary antibody (B) and visualized by confocal miscroscopy (100X). White arrows indicate areas in which Nox1- (lower left panels) and F actin in (A) or cortactin in (B) (middle panels) colocalize in structures identified as invadopodia (merge in yellow). Scale bars, 5μm. One representative picture from three separate experiments is shown. (C) and (D) Nox1 localizes to cortactin-rich structures capable of degrading the ECM in DLD1 cells. DLD1 cells were transfected with SrcYF and with RFP-tagged Nox1 or RFP empty vector in (C) and with GFP-tagged Nox1 or GFP empty vector in (D). After 24hrs, cells were trypsinized and plated on FITC-labeled gelatin-coated coverslips. 48hrs later, cells were fixed in 4%PFA in (C) or methanol in (D) and stained with mouse cortactin antibody, followed by anti mouse Alexa-Fluor 647-conjugated secondary antibody in (C) and (D) and with rabbit polyclonal GFP antibody, followed by anti-rabbit Alexa-Fluor 568-conjugated secondary antibody in (D). Successively, cells were visualized by confocal miscroscopy (60X). White arrows indicate areas in which Nox1 (in red) and cortactin (in green) colocalize in invadopodia (yellow in the merge) capable of degrading the ECM (in blue). Scale bars, 4μm. One representative image from three separate experiments is shown.

Figure 6. The overexpression of NoxO1 in DLD1 cells reduces invadopodia formation and ECM degradation

(A) and (B) NoxO1 overexpression reduces invadopodia formation in a concentration-dependent manner in DLD1 cells. DLD1 cells were transfected with SrcYF, GFP-tagged Nox1, and with increasing amounts of NoxO1 (1x or 3x) or with equal amount of empty vector (mock). After 48hrs, cells were fixed in 4%PFA, stained with Alexa-Fluor-568 phalloidin in (A) or antibody followed by Alexa-Fluor 568-conjugated secondary antibody in (B) and visualized by confocal miscroscopy (100X). In the upper panels, the white arrows indicate areas in which Nox1- (left panels) and F actin in (A) or cortactin in (B) (middle panels) colocalize in invadopodia (merge in yellow). Scale bars, 5μm. One representative image from three separate experiments is shown. (C) The overexpression of NoxO1 in DLD1 cells reduces ECM degradation. DLD1 cells were transfected with SrcYF, RFP-tagged Nox1 and with NoxO1 (3x) or with equal amount of empty vector (mock). 24hrs later, cells were trypsinized and plated on FITC-labeled gelatin-coated coverslips, and after 48hrs the cells were fixed in 4%PFA and visualized by epifluorescence microscopy (40X). In the left panel, the white arrows indicate areas in which RFP-tagged Nox1-expressing cells (in red) degrade the ECM (in green). Scale bars, 45μm. One representative image from three separate experiments is shown. (D) The overexpression of NoxO1 in DLD1 cells reduces the number of ROS-positive invadopodia-like structures. DLD1 cells were transfected with SrcYF and with NoxO1 or empty vector (mock). 72hrs later, cells were incubated with 2.5μM of the ROS-sensitive probe PY1-AM in HBSS for 30 minutes at 37C (see Methods) and visualized by confocal microscopy (100X). One representative image from three separate experiments is shown. Scale bars, 5μm. (E) Quantifications of experiments illustrated in (A) to (D). In the first two panels quantification from three independent biological experiments shown in (A) and (B) is given: the number of Nox1/phalloidin positive-structures in (A) or Nox1/cortactin positive-structures in (B) was counted and averaged from 25 cells for each experiment. Error bars represent SEM. *p<0.008 **p<0.002. In the third panel, quantification from three independent biological experiments as shown in (C): for each experiment, the total degradation area was obtained as sum of degradation areas calculated using Metamorph software from 25 images and reported as percentage (mock set as 100%). In the graph, error bars represent SEM. *p<0.02. In the right panel, quantification from three independent biological experiments, as shown in (D): the number of ROS-positive structures was counted and averaged from 10 pictures for each experiment. Error bars represent SEM. *p<0.005