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Nitric oxide-induced epidermal growth factor-dependent phosphorylations in A431 tumour cells
María J Ruano1, Silvia Hernández-Hernando, Amparo Jiménez
1Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Spain.
Abstract:
Nitric oxide (NO*) strongly inhibits the proliferation of human A431 tumour cells. It also inhibits tyrosine phosphorylation of a 170-kDa band corresponding to the epidermal growth factor receptor (EGFR) and induces the phosphorylation at tyrosine residue(s) of a 58-kDa protein which we have denoted NOIPP-58 (nitric oxide-induced 58-kDa phosphoprotein). The NO*-induced phosphorylation of NOIPP-58 is strictly dependent on the presence of EGF. Phosphorylation of NOIPP-58 and inhibition of the phosphorylation of the band corresponding to EGFR are both cGMP-independent processes. We also demonstrate that the p38 mitogen-activated protein kinase (p38MAPK) pathway is activated by NO* in the absence and presence of EGF, whereas the activity of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and the c-Jun N-terminal kinase 1/2 (JNK1/2) pathways are not significantly affected or are slightly decreased, respectively, on addition of this agent. Moreover, we show that the p38MAPK inhibitor, SB202190, induces rapid vanadate/peroxovanadate-sensitive dephosphorylation of prephosphorylated EGFR and NOIPP-58. We propose that the dephosphorylation of both NOIPP-58 and EGFR are mediated by a p38MAPK-controlled phosphotyrosine-protein phosphatase (PYPP). Activation of the p38MAPK pathway during nitrosative stress probably prevents the operation of this PYPP, allowing NOIPP-58, and in part EGFR, to remain phosphorylated and therefore capable of generating signalling events.
Insights
Nitric oxide (NO) inhibits tumor cell growth by affecting protein phosphorylation. NO activates the p38 MAPK pathway, influencing epidermal growth factor receptor (EGFR) and NOIPP-58 phosphorylation.
Area of Science:
- Cellular signaling pathways
- Cancer biology
- Molecular mechanisms of cell proliferation
Background:
- Nitric oxide (NO) is a key signaling molecule with diverse biological roles.
- Epidermal growth factor receptor (EGFR) signaling is crucial for cell growth and is often dysregulated in cancer.
- Protein phosphorylation plays a critical role in regulating cellular processes, including proliferation and signal transduction.
Purpose of the Study:
- To investigate the effects of nitric oxide (NO) on human A431 tumor cell proliferation.
- To elucidate the molecular mechanisms by which NO influences protein tyrosine phosphorylation, particularly of EGFR and a novel 58-kDa phosphoprotein (NOIPP-58).
- To determine the role of specific mitogen-activated protein kinase (MAPK) pathways in NO-mediated signaling.
Main Methods:
- Cell proliferation assays using human A431 tumor cells.
- Western blotting to detect tyrosine phosphorylation of EGFR and NOIPP-58.
- Analysis of MAPK pathway activation (p38MAPK, ERK1/2, JNK1/2) using specific inhibitors and activators.
- Investigation of the role of phosphotyrosine-protein phosphatase (PYPP) in NO-induced signaling.
Main Results:
- NO strongly inhibited A431 tumor cell proliferation.
- NO induced tyrosine phosphorylation of NOIPP-58 and inhibited EGFR phosphorylation in a cGMP-independent manner.
- The p38 MAPK pathway was activated by NO, while ERK1/2 and JNK1/2 pathways were not significantly affected or were decreased.
- Inhibition of p38MAPK led to dephosphorylation of EGFR and NOIPP-58, suggesting a role for a p38MAPK-controlled phosphatase.
Conclusions:
- NO exerts anti-proliferative effects on tumor cells by modulating protein phosphorylation.
- The p38MAPK pathway is a key mediator of NO's effects on EGFR and NOIPP-58 phosphorylation.
- A p38MAPK-regulated phosphotyrosine-protein phosphatase (PYPP) is involved in the dephosphorylation of EGFR and NOIPP-58, with its activity potentially inhibited by NO-induced p38MAPK activation.