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Publication
S-nitrosylation of endogenous protein tyrosine phosphatases in endothelial
insulin signaling.
Authors Hsu MF, Pan KT, Chang FY, Khoo KH, Urlaub H, Cheng CF, Chang GD, Haj FG, Meng TC
Submitted By Submitted Externally on 5/1/2017
Status Published
Journal Free radical biology & medicine
Year 2016
Date Published 10/1/2016
Volume : Pages 99 : 199 - 213
PubMed Reference 27521458
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.




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