title: S-nitrosylation of endogenous protein tyrosine phosphatases in endothelial insulin signaling.
abstract: Nitric oxide (NO) exerts its biological function through S-nitrosylation of cellular proteins. Due to the labile nature of this modification under physiological condition, identification of S-nitrosylated residue in enzymes involved in signaling regulation remains technically challenging. The present study investigated whether intrinsic NO produced in endothelium-derived MS-1 cells response to insulin stimulation might target endogenous protein tyrosine phosphatases (PTPs). For this, we have developed an approach using a synthetic reagent that introduces a phenylacetamidyl moiety on S-nitrosylated Cys, followed by detection with anti-phenylacetamidyl Cys (PAC) antibody. Coupling with sequential blocking of free thiols with multiple iodoacetyl-based Cys-reactive chemicals, we employed this PAC-switch method to show that endogenous SHP-2 and PTP1B were S-nitrosylated in MS-1 cells exposed to insulin. The mass spectrometry detected a phenylacetamidyl moiety specifically present on the active-site Cys463 of SHP-2. Focusing on the regulatory role of PTP1B, we showed S-nitrosylation to be the principal Cys reversible redox modification in endothelial insulin signaling. The PAC-switch method in an imaging format illustrated that a pool of S-nitrosylated PTP1B was colocalized with activated insulin receptor to the cell periphery, and that such event was endothelial NO synthase (eNOS)-dependent. Moreover, ectopic expression of the C215S mutant of PTP1B that mimics the active-site Cys215 S-nitrosylated form restored insulin responsiveness in eNOS-ablated cells, which was otherwise insensitive to insulin stimulation. This work not only introduces a new method that explores the role of physiological NO in regulating signal transduction, but also highlights a positive NO effect on promoting insulin responsiveness through S-nitrosylation of PTP1B's active-site Cys215.
authors: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors: Ming-Fo
authors: MF
authors: Hsu
authors: Bioanalytical Mass Spectrometry Group, Max Plank Institute for Biophysical Chemistry, Göttingen, Germany.
authors: Kuan-Ting
authors: KT
authors: Pan
authors: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan.
authors: Fan-Yu
authors: FY
authors: Chang
authors: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan.
authors: Kay-Hooi
authors: KH
authors: Khoo
authors: Bioanalytical Mass Spectrometry Group, Max Plank Institute for Biophysical Chemistry, Göttingen, Germany; Bioanalytics Research Group, Department of Clinical Chemistry, University Medical Center, Göttingen, Germany.
authors: Henning
authors: H
authors: Urlaub
authors: Department of Medical Research, Tzu Chi University and Department of Pediatrics, Tzu Chi General Hospital, Hualien, Taiwan; Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
authors: Ching-Feng
authors: CF
authors: Cheng
authors: Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan. Electronic address: gdchang@ntu.edu.tw.
authors: Geen-Dong
authors: GD
authors: Chang
authors: Department of Nutrition, University of California Davis, Davis, CA, USA. Electronic address: fghaj@ucdavis.edu.
authors: Fawaz G
authors: FG
authors: Haj
authors: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan. Electronic address: tcmeng@gate.sinica.edu.tw.
authors: Tzu-Ching
authors: TC
authors: Meng
mesh_terms: D000083:Acetanilides / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D019556:COS Cells; D020134:Catalytic Domain; D002460:Cell Line; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D007202:Indicators and Reagents / Q000737:chemistry; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D052250:Nitric Oxide Synthase Type III / Q000172:deficiency / Q000235:genetics; D009603:Nitroso Compounds / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000235:genetics / Q000378:metabolism*; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000235:genetics / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000083:Acetanilides; D000906:Antibodies; D007202:Indicators and Reagents; D007328:Insulin; D009603:Nitroso Compounds; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D011972:Receptor, Insulin; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; C516609:Ptpn1 protein, mouse; D003545:Cysteine
keywords: Endothelial cell; Insulin signaling; New method; Nitric oxide; PTP1B; S-nitrosylation; SHP-2