Structural and biochemical studies of p21Ras S-nitrosylation and nitric oxide-mediated guanine nucleotide exchange

Our studies show that stable S-nitrosylation of Ras at Cys-118, does not affect the structure of Ras, its association with the Ras-binding domain of Raf, or guanine nucleotide exchange rates relative to non-nitrosylated Ras

J. G. Williams; K. Pappu; S. L. Campbell


Scholarcy highlights

  • N early 20 years ago, nitric oxide was identified as a vaso-relaxation factor and was found to mediate its effects on smooth muscle by coordinating the heme moiety of guanylate cyclase, leading to enhanced production of cGMP
  • To further elucidate how treatment of Ras with NO donating agents leads to Ras activation in vivo, we conducted structural and biochemical studies on S-nitrosylated Ras
  • Our studies revealed that stable S-nitrosylation of Ras at Cys-118 does not cause any significant perturbations in the secondary or tertiary structure of Ras, and does not influence Ras guanine nucleotide exchange in the presence or absence of the Ras guanine-nucleotide exchange factors, SOS
  • The site of S-nitrosylation is removed from the effector binding region ; it was not clear whether more subtle structural alterations in Ras resulting from S-nitrosylation might affect effector binding, because previous studies indicated that Ras nitrosylation leads to increased association of Ras to the Ras effectors Raf-1 and phosphatidylinositol-3 kinase
  • Our results indicate that neither Raf-RBD binding nor SOS-mediated GNE of Ras is altered by stable S-nitrosylation of Ras, we cannot exclude the possibility that other GEF(s), guanosine triphosphatases-activating protein(s), or effectors may be altered in their abilities to recognize S-nitrosylated Ras relative to its non-nitrosylated counterpart
  • Whereas our structural and biochemical studies indicate that the end-product of S-nitrosylation of Ras does not alter Ras structure, GNE or Raf-RBD binding, the data set forth in this paper indicate that an intermediate of the nitrosylation reaction, rather than the R-S-NO end product, alters Ras structural elements and biochemical properties
  • In the case of Cys-nitric oxide, the affect seems to be specific because neither nitrite, nitrate, nor cysteine promotes guanine nucleotide exchange exchange on Ras. Consistent with these observations, we have found that NO gas, Cys-NO, and GS-NO can modify Ras through a radical mechanism, where formation of a Ras-radical intermediate reduces the affinity of Ras for its guanine nucleotide substrate, leading to enhanced guanine nucleotide dissociation

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