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.
Highlights
We reveal that inducible nitric-oxide synthase, an enzyme implicated in cancer and inflammation, is a downstream mediator of activated Src
INOS Is Phosphorylated on a Tyrosine Residue—Prior studies suggested that inducible nitric-oxide synthase (iNOS) is subject to tyrosine phosphorylation [14, 15]
We examined iNOS following its transfection in HEK293 cells
Summary
Human iNOS, purified by immunoprecipitation from iNOS-transfected HEK293 cells, was incubated in the presence or absence of Src (100 ng) and ATP (100 M) for 20 min at 30 °C and analyzed by Western blotting using iNOS or pY antibodies. Src inhibition markedly reduced tyrosinephosphorylated iNOS, as detected by co-immunoprecipitation (Fig. 2E), confirming the role of Src in iNOS tyrosine phosphorylation. We examined iNOS tyrosine phosphorylation in HEK293 cells transfected with wild-type iNOS or with the Y1055F iNOS mutant.
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