Format

Send to

Choose Destination
Free Radic Biol Med. 2016 Oct;99:199-213. doi: 10.1016/j.freeradbiomed.2016.08.012. Epub 2016 Aug 10.

S-nitrosylation of endogenous protein tyrosine phosphatases in endothelial insulin signaling.

Author information

1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
2
Bioanalytical Mass Spectrometry Group, Max Plank Institute for Biophysical Chemistry, Göttingen, Germany.
3
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan.
4
Bioanalytical Mass Spectrometry Group, Max Plank Institute for Biophysical Chemistry, Göttingen, Germany; Bioanalytics Research Group, Department of Clinical Chemistry, University Medical Center, Göttingen, Germany.
5
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.
6
Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan. Electronic address: gdchang@ntu.edu.tw.
7
Department of Nutrition, University of California Davis, Davis, CA, USA. Electronic address: fghaj@ucdavis.edu.
8
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.

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.

KEYWORDS:

Endothelial cell; Insulin signaling; New method; Nitric oxide; PTP1B; S-nitrosylation; SHP-2

[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center