Src kinase-mediated phosphorylation stabilizes inducible nitric-oxide synthase in normal cells and cancer cells

Abstract

Src kinases are key regulators of cellular proliferation, survival, motility, and invasiveness. They play important roles in the regulation of inflammation and cancer. Overexpression or hyperactivity of c-Src has been implicated in the development of various types of cancer, including lung cancer. Src inhibition is currently being investigated as a potential therapy for non-small cell lung cancer in Phase I and II clinical trials. The mechanisms of Src implication in cancer and inflammation are linked to the ability of activated Src to phosphorylate multiple downstream targets that mediate its cellular effector functions. In this study, we reveal that inducible nitric-oxide synthase (iNOS), an enzyme also implicated in cancer and inflammation, is a downstream mediator of activated Src. We elucidate the molecular mechanisms of the association between Src and iNOS in models of inflammation induced by lipopolysaccharide and/or cytokines and in cancer cells and tissues. We identify human iNOS residue Tyr(1055) as a target for Src-mediated phosphorylation. These results are shown in normal cells and cancer cells as well as in vivo in mice. Importantly, such posttranslational modification serves to stabilize iNOS half-life. The data also demonstrate interactions and co-localization of iNOS and activated Src under inflammatory conditions and in cancer cells. This study demonstrates that phosphorylation of iNOS by Src plays an important role in the regulation of iNOS and nitric oxide production and hence could account for some Src-related roles in inflammation and cancer.

Figures

FIGURE 1.

iNOS is tyrosine-phosphorylated. A, HEK293 cells were transfected for 24 h with a plasmid encoding cDNA of human iNOS. RAW264.7 cells were stimulated for 12 h by 100 ng/ml LPS. RT4 cells, A549 cells, and primary NHBE cells were stimulated for 18 h by a cytokine mixture of interferon-γ (100 units/ml; 500 units/ml for A549), interleukin-1β (0.5 ng/ml), and tumor necrosis factor-α (10 ng/ml). Cell lysates were subjected to immunoprecipitation (IP) with iNOS antibody followed by Western blot analysis using antibodies against phosphotyrosine (pY) or iNOS. B, immunoprecipitated iNOS, from RAW264.7 cells, was incubated with 10 units of leukocyte common antigen-related protein-tyrosine phosphatase for 30 min prior to Western blot analysis. C, prior to immunoprecipitation, iNOS-transfected HEK293 cells or LPS-stimulated RAW264.7 cells were incubated with 10 mm sodium o-van for 4 h or 1 h, respectively. Cytokine-stimulated NHBE cells were incubated with 100 μm PV for 30 min prior to immunoprecipitation.

FIGURE 2.

iNOS is regulated by Src-dependent tyrosine phosphorylation. A and B, exogenous expression of Src kinase increases iNOS steady-state level. HEK293 cells were co-transfected, in a 1:1 molar ratio, with plasmids encoding cDNA of human iNOS and with either Src kinase or LacZ, as a control. A, 24 h after transfection, cells were lysed, and aliquots of lysates were subjected to Western blotting with antibodies against iNOS, Src, or β-actin. B, NO production by iNOS was evaluated by measuring nitrite accumulation in culture media. Data represent mean ± S.D., n = 6. *, p < 0.05 compared with control condition. C, Src induces iNOS phosphorylation in cultured cells. Aliquots of lysates were also used for immunoprecipitation (IP) with iNOS antibody followed by Western blotting using phosphotyrosine (pY) antibody. To verify the stringency of immunoprecipitation, HEK293 cells that do not express iNOS were used as a negative control. D, Src interacts with iNOS. The remainder of cell lysates was subjected to immunoprecipitation with Src antibody and analyzed by Western blotting using antibodies against Src or iNOS. To verify the stringency of immunoprecipitation, experiments included samples in which immunoprecipitation antibody was replaced with irrelevant antibody (anti-IκB-β, middle lane). E and F, iNOS tyrosine phosphorylation is attenuated by Src inhibition. E, HEK293 cells were co-transfected as in A, except that the Src kinase inhibitor PP2 (20 μm) or its vehicle dimethyl sulfoxide (DMSO) was added to culture media 6 h prior to cell lysis. F, HEK293 cells were transfected with human iNOS cDNA. Twenty-four hours after transfection and prior to cell lysis, cells were sequentially incubated in the presence or absence of PP2 (20 μm; 30 min) followed by the tyrosine phosphatase inhibitor PV (100 μm for 30 min). Cell lysates were subjected to immunoprecipitation with iNOS antibody followed by Western blotting with pY antibody. G, Src induces iNOS phosphorylation in vitro. Human iNOS, purified by immunoprecipitation from iNOS-transfected HEK293 cells, was incubated in the presence or absence of Src (100 ng) and ATP (100 μm) for 20 min at 30 °C and then analyzed by Western blotting using iNOS or pY antibodies.

FIGURE 3.

iNOS is phosphorylated on residue Tyr1055. HEK293 cells were transfected for 24 h with a plasmid encoding cDNA of human iNOS. RAW264.7 cells were stimulated for 12 h by 100 ng/ml LPS. RT4 cells, A549 cells, and primary NHBE were stimulated for 18 h by a cytokine mixture of interferon-γ, interleukin-1β, and tumor necrosis factor-α. A, aliquots of cell lysates were subjected to Western blot analysis using iNOS or β-actin antibodies. B, the remainder of cell lysates was subjected to immunoprecipitation (IP) with an antibody against Tyr1055-phosphorylated iNOS (pY1055) followed by Western blotting using iNOS antibodies. To verify the stringency of immunoprecipitation, experiments included samples in which immunoprecipitation antibody was replaced with irrelevant antibody (anti-IκB-β). C, mice were injected intraperitoneally with either LPS (1 mg/kg) or vehicle (phosphate-buffered saline) and sacrificed 18 h later, and their lungs were dissected. Tissue lysates were subjected to Western blot analysis with antibodies against iNOS or β-actin (upper panel). Another part of lysates was subjected to immunoprecipitation with pY1055 antibody. Immunoprecipitates were analyzed by Western blotting using anti-iNOS antibodies (lower panel).

FIGURE 4.

Characterization of Human iNOS Tyr1055 phosphorylation. A and B, Y1055F mutation reduces iNOS phosphorylation. HEK293 cells were either not transfected or were transfected for 24 h with plasmids encoding cDNAs of wild-type (WT) iNOS or iNOS mutant Y1055F. Cell lysates were subjected to immunoprecipitation (IP) with Tyr1055-phosphoryated iNOS (pY1055) (A) or iNOS (B) antibodies. Aliquots of immunoprecipitation were subjected to Western blotting using iNOS (A) or pY (B) antibodies. C and D, o-van increases iNOS Tyr1055 phosphorylation. C, HEK293 cells were transfected, for 24 h, with a plasmid encoding iNOS cDNA and then incubated in the presence or absence of 10 mm o-van for 4 h. Cells were lysed and subjected to immunoprecipitation using pY1055 antibody followed by Western blotting using iNOS or pY antibodies. To verify the stringency of immunoprecipitation, experiments included samples where immunoprecipitation antibody was replaced with irrelevant antibody (anti-IκB-β), or immunoprecipitation was done on lysates of HEK293 cells not transfected with iNOS. D, HEK293 cells were transfected as in A and then incubated in the presence or absence of 10 mm o-van for 4 h. Cells were lysed and subjected to immunoprecipitation with pY1055 antibody followed by Western blotting with iNOS antibody. E, Src kinase increases iNOS Tyr1055 phosphorylation. HEK293 cells were co-transfected for 24 h, in a 1:1 molar ratio, with plasmids encoding human iNOS and either Src or LacZ as a control. Cell lysates were used for immunoprecipitation with pY1055 antibody followed by Western blotting with iNOS antibody.

FIGURE 5.

Y1055F mutation reduces iNOS steady-state level. HEK293 cells were transfected with a plasmid encoding cDNA of wild-type (WT) iNOS or the iNOS Y1055F mutant. A, 24 h after transfection, cell lysates were analyzed by Western blotting using iNOS or β-actin antibodies. B and C, iNOS activity was evaluated in cell lysates (B) and by measuring nitrite accumulation in culture media (C). D, 24 h after transfection, the proteasomal inhibitor MG132 (10 μm) or its vehicle (dimethyl sulfoxide) was added to cells for 18 h. E, 24 h after transfection, cells were pulsed with [35S]methionine/cysteine for 1 h and chased with unlabeled media at various time points. iNOS was immunoprecipitated with anti-iNOS antibody. Eluted proteins were analyzed by SDS-PAGE. Bands, representing 35S-labeled iNOS, were quantitated to calculate iNOS half-life. Data represent mean ± S.D. *, p < 0.05; **, p < 0.001.

FIGURE 6.

Activation of Src kinase by EGF increases iNOS Tyr1055 phosphorylation. A, HeLa cells were transfected for 24 h with a plasmid encoding cDNA of human iNOS or LacZ, as a control. Cells were then incubated for 20 min in the presence or absence of 100 ng/ml EGF before lysis. Cell lysates were subjected to Western blotting using antibodies against iNOS, Src, p(416)Src, or β-actin. B, aliquots of cells lysates were subjected to immunoprecipitation (IP) with pY1055 iNOS antibody. Immunoprecipitates were analyzed with Western blotting using iNOS antibody.

FIGURE 7.

Co-localization of iNOS and active Src in airway epithelium of mouse lung tissue. Mice were intraperitoneally injected with either phosphate-buffered saline (vehicle only; A) or LPS (1 mg/kg; B) for 18 h before they were killed. Frozen sections of lung tissues were fixed and immunolabeled by anti-iNOS (mouse) and anti-p(416)Src (rabbit) antibodies followed by goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red), respectively. Cells were stained with 4′,6-diamidino-2-phenylindol dihydrochloride (DAPI) to visualize nuclei (blue). Scale bars, 10 μm.

FIGURE 8.

Co-localization of iNOS and active Src in airway epithelium of NSC lung cancer tissue. A, frozen sections of NSC lung cancer tissue were fixed and immunolabeled by iNOS (mouse) and p(416)Src (rabbit) antibodies followed by goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red), respectively. Cells were stained with DAPI to visualize nuclei (blue). B, H&E staining of the tissue. Arrows denote apical part of the airway epithelial cells. C, higher magnification of section indicated by box in A. Arrows denote apical part of the airway epithelial cells. D, negative control. Tissues were evaluated as in A, except primary antibodies were omitted. Scale bars, 10 μm.

FIGURE 9.

Proposed model of Src-mediated phosphorylation regulation of iNOS. Src interacts with and phosphorylates iNOS on Tyr1055 and thus stabilizes active iNOS. Dephosphorylation of Tyr1055 triggers targeting of iNOS for proteasomal degradation. These results are further illustrated in the iNOS Y1055F mutant, which is rapidly degraded by the proteasome. P, phosphate group; PTP, protein-tyrosine phosphatase.