4207

In previous work (Yakovlev et al, Biochemistry, Oct 23; 46(42):11671-83, 2007) we demonstrated that ionizing radiation (IR) in the therapeutic dose range stimulates NF-kappaB activity by a mechanism in which IkappaB-alpha tyrosine 181 is nitrated as a consequence of constitutive NO• synthase activation, leading to dissociation of intact IkappaB-alpha from NF-kappaB. We have also identified a number of other proteins that are nitrated after radiation including the tumor suppressor protein, p53. Previous studies demonstrated that activated NO• synthase or nitric oxide donors nitrate p53 resulting in an increase of p53-DNA binding activity. However, the underlying mechanism of NO-dependent p53 activation and whether tyrosine nitration is involved remain unknown. We show that low doses of IR lead to p53 tyrosine nitration along with its rapid activation. Moreover, nitration of p53 promotes tetramerization of this protein in a dose-dependent manner. Mass Spectrometry analysis revealed that p53 nitration occurs on tyrosine 107 in the DNA binding domain, and tyrosine 327 in the tetramerization domain. We tested whether nitration results in conformational changes in p53 tetramerization domain. Cross-linking experiments support the argument that nitration can lead to an affinity increase between p53-monomers and, as a result, rapid p53 tetramerization - a critical step in p53 activation.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA