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Binding of active Ras and its mutants to the Ras binding domain of PI-3-kinase: A quantitative approach to KD measurements.
Fleming, Ian R; Hannan, Jonathan P; Swisher, George Hayden; Tesdahl, Corey D; Martyr, Justin G; Cordaro, Nicholas J; Erbse, Annette H; Falke, Joseph J.
Afiliación
  • Fleming IR; Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, CO, 80309-0596, USA.
  • Hannan JP; Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, CO, 80309-0596, USA.
  • Swisher GH; Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, CO, 80309-0596, USA.
  • Tesdahl CD; Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, CO, 80309-0596, USA.
  • Martyr JG; Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, CO, 80309-0596, USA.
  • Cordaro NJ; Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, CO, 80309-0596, USA.
  • Erbse AH; Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, CO, 80309-0596, USA.
  • Falke JJ; Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, CO, 80309-0596, USA. Electronic address: falke@colorado.edu.
Anal Biochem ; 663: 115019, 2023 02 15.
Article en En | MEDLINE | ID: mdl-36526022
ABSTRACT
Ras family GTPases (H/K/N-Ras) modulate numerous effectors, including the lipid kinase PI3K (phosphatidylinositol-3-kinase) that generates growth signal lipid PIP3 (phosphatidylinositol-3,4,5-triphosphate). Active GTP-Ras binds PI3K with high affinity, thereby stimulating PIP3 production. We hypothesize the affinity of this binding interaction could be significantly increased or decreased by Ras mutations at PI3K contact positions, with clinical implications since some Ras mutations at PI3K contact positions are disease-linked. To enable tests of this hypothesis, we have developed an approach combining UV spectral deconvolution, HPLC, and microscale thermophoresis to quantify the KD for binding. The approach measures the total Ras concentration, the fraction of Ras in the active state, and the affinity of active Ras binding to its docking site on PI3K Ras binding domain (RBD) in solution. The approach is illustrated by KD measurements for the binding of active H-Ras and representative mutants, each loaded with GTP or GMPPNP, to PI3Kγ RBD. The findings demonstrate that quantitation of the Ras activation state increases the precision of KD measurements, while also revealing that Ras mutations can increase (Q25L), decrease (D38E, Y40C), or have no effect (G13R) on PI3K binding affinity. Significant Ras affinity changes are predicted to alter PI3K regulation and PIP3 growth signals.
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Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Proteínas ras / Fosfatidilinositol 3-Quinasas Tipo de estudio: Prognostic_studies Idioma: En Revista: Anal Biochem Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Proteínas ras / Fosfatidilinositol 3-Quinasas Tipo de estudio: Prognostic_studies Idioma: En Revista: Anal Biochem Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos