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

Ming Fo Hsu, Kuan Ting Pan, Fan Yu Chang, Kay Hooi Khoo, Henning Urlaub, Ching Feng Cheng, Geen Dong Chang, Fawaz Haj, Tzu Ching Meng

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)199-213
Number of pages15
JournalFree Radical Biology and Medicine
StatePublished - Oct 1 2016


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

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)


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