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Nitric oxide and superoxide inhibit platelet-derived growth factor receptor phosphotyrosine phosphatases
D Callsen1, K B Sandau, B Brüne
1University of Erlangen-Nürnberg, Faculty of Medicine, Department of Medicine, Germany.
Abstract:
Platelet derived growth factor receptor (PDGFR) became tyrosine autophosphorylated in rat mesangial cells shortly after platelet derived growth factor (PDGF) ligation in a tyrosine kinase inhibitor (tyrphostin AG 1296) sensitive manner. Ligand-independent, massive tyrosine PDGFR phosphorylation was achieved by diverse NO releasing compounds. Phosphorylation was slow compared to PDGF, revealed a concentration- and time-dependency, and was not mimicked by lipophilic cyclic-GMP analogues. Interleukin-1 beta/cAMP activated mesangial cells released NO and in turn showed PDGFR phosphorylation. A NO-synthase involvement was assured by L-NG-nitroarginine methyl ester inhibition. PDGFR phosphorylation was also achieved by the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. NO- and O2(.-)-evoked PGDFR phosphorylation was N-acetylcysteine reversible. Cell free dephosphorylation assays revealed PDGFR dephosphorylation by tyrosine phosphatases. Receptor dephosphorylation by cytosolic phosphatases was completed within 30 min and was sensitive to the readdition of NO donors or orthovanadate. In addition, phosphatase activity determined in a direct dephosphorylation assay using the substrate para-nitrophenyl phosphate was attenuated by NO or vanadate. We conclude that cytosolic protein tyrosine phosphatases are targeted by exogenously supplied or endogenously generated NO in mesangial cells. Radical (NO. or O2.-) formation shifts the phosphorylation--dephosphorylation equilibrium towards phosphorylation, thus integrating redox-mediated responses into established signal transducing pathways.
Insights
Nitric oxide (NO) triggers tyrosine phosphorylation of platelet-derived growth factor receptor (PDGFR) in mesangial cells. This NO-mediated PDGFR phosphorylation involves cytosolic protein tyrosine phosphatases, shifting the balance toward phosphorylation.
Area of Science:
- Cellular signaling
- Biochemistry
- Renal physiology
Background:
- Platelet-derived growth factor receptor (PDGFR) signaling is crucial in mesangial cell function.
- The role of nitric oxide (NO) in regulating PDGFR phosphorylation in mesangial cells is not fully understood.
Purpose of the Study:
- To investigate the effect of nitric oxide (NO) on PDGFR tyrosine autophosphorylation in rat mesangial cells.
- To elucidate the mechanisms underlying NO-induced PDGFR phosphorylation and dephosphorylation.
Main Methods:
- Treatment of rat mesangial cells with NO-releasing compounds and platelet-derived growth factor (PDGF).
- Assessment of PDGFR tyrosine phosphorylation using tyrosine kinase inhibitors and NO-synthase inhibitors.
- Evaluation of tyrosine phosphatase activity in cell-free dephosphorylation assays.
Main Results:
- NO donors induced ligand-independent PDGFR tyrosine phosphorylation in a time- and concentration-dependent manner.
- Endogenous NO release, stimulated by Interleukin-1 beta/cAMP, also led to PDGFR phosphorylation.
- NO and superoxide radicals (O2.-) inhibited tyrosine phosphatase activity, favoring PDGFR phosphorylation.
Conclusions:
- Cytosolic protein tyrosine phosphatases are targeted by NO in mesangial cells.
- NO and related radicals shift the phosphorylation-dephosphorylation balance of PDGFR towards phosphorylation.
- Redox-mediated signaling integrates into established PDGFR signal transduction pathways.