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RhoA inactivation by S-nitrosylation regulates vascular smooth muscle contractive signaling.

Publication ,  Journal Article
Lin, L; Xu, C; Carraway, MS; Piantadosi, CA; Whorton, AR; Li, S
Published in: Nitric Oxide
April 1, 2018

S-nitrosothiols derived from nitric oxide are known to regulate cell signaling through thiol modification. Since small G protein RhoA contains cysteine residues in the GTP-binding domain which is critical for its function, modification these thiols may alter RhoA activity and lead to changes in the downstream signaling such as myosin light chain phosphorylation. However, it is still unclear that if RhoA activity and its downstream signals might be modulated by S-nitrosothiols and if the two cysteine residues located in the GTP-binding domain are critical for the regulation. In this study we show that S-nitroso-L-cysteine (CSNO) blocked RhoA activation as determined by either GDP/GTP exchange, active RhoA binding to rhotekin or RhoA translocation. CSNO was shown to lead to RhoA nitrosylation and RhoA thiol oxidation status was found to be consistent with loss of its activity. Mutation of all 6 single cysteine residues to serine showed that purified recombinant C20S mutant and C26/20S mutant were resistant to CSNO, but interestingly, in the intact cells only the double C16/20S mutant was resistant to CSNO. Moreover, inhibition of RhoA activation led to Rho-kinase inhibition and inhibition of Rho pathway signaling by CSNO. In both smooth muscle cells and aortic tissue, the outcome was inhibition of agonist-stimulated MYPT1 phosphorylation and reduced levels of myosin light chain phosphorylation. These effects of CSNO on MYPT1 and myosin light chain phosphorylation appear to be cGMP-independent since they were unaffected by inhibition of guanylyl cyclase. In contrast to CSNO, spermine NONOate did not alter RhoA GDP/GTP exchange and the effects of this compound on myosin light chain phosphorylation were blocked by guanylyl cyclase inhibition. And importantly, in C16/20S overexpressed smooth muscle cells, MYPT1 phosphorylation was resistant to the inhibitory effect of CSNO. Together, these data suggest that S-nitrosothiols regulate myosin light chain phosphorylation by inhibiting RhoA/Rho-kinase signaling through modification of RhoA cysteine residues at 16 and 20 in its GTP-binding domain, which might be an important therapeutic target for diseases with imbalanced vascular resistance.

Duke Scholars

Published In

Nitric Oxide

DOI

EISSN

1089-8611

Publication Date

April 1, 2018

Volume

74

Start / End Page

56 / 64

Location

United States

Related Subject Headings

  • rhoA GTP-Binding Protein
  • Signal Transduction
  • S-Nitrosothiols
  • Rats
  • Nitric Oxide
  • Muscle, Smooth, Vascular
  • Muscle Contraction
  • Humans
  • HEK293 Cells
  • Biochemistry & Molecular Biology
 

Citation

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Lin, L., Xu, C., Carraway, M. S., Piantadosi, C. A., Whorton, A. R., & Li, S. (2018). RhoA inactivation by S-nitrosylation regulates vascular smooth muscle contractive signaling. Nitric Oxide, 74, 56–64. https://doi.org/10.1016/j.niox.2018.01.007
Lin, Li, Chang Xu, Martha S. Carraway, Claude A. Piantadosi, A Richard Whorton, and Sheng Li. “RhoA inactivation by S-nitrosylation regulates vascular smooth muscle contractive signaling.Nitric Oxide 74 (April 1, 2018): 56–64. https://doi.org/10.1016/j.niox.2018.01.007.
Lin L, Xu C, Carraway MS, Piantadosi CA, Whorton AR, Li S. RhoA inactivation by S-nitrosylation regulates vascular smooth muscle contractive signaling. Nitric Oxide. 2018 Apr 1;74:56–64.
Lin, Li, et al. “RhoA inactivation by S-nitrosylation regulates vascular smooth muscle contractive signaling.Nitric Oxide, vol. 74, Apr. 2018, pp. 56–64. Pubmed, doi:10.1016/j.niox.2018.01.007.
Lin L, Xu C, Carraway MS, Piantadosi CA, Whorton AR, Li S. RhoA inactivation by S-nitrosylation regulates vascular smooth muscle contractive signaling. Nitric Oxide. 2018 Apr 1;74:56–64.
Journal cover image

Published In

Nitric Oxide

DOI

EISSN

1089-8611

Publication Date

April 1, 2018

Volume

74

Start / End Page

56 / 64

Location

United States

Related Subject Headings

  • rhoA GTP-Binding Protein
  • Signal Transduction
  • S-Nitrosothiols
  • Rats
  • Nitric Oxide
  • Muscle, Smooth, Vascular
  • Muscle Contraction
  • Humans
  • HEK293 Cells
  • Biochemistry & Molecular Biology