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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

Ian R Fleming1, Jonathan P Hannan1, George Hayden Swisher1

  • 1Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, CO, 80309-0596, USA.

Analytical Biochemistry
|December 16, 2022
PubMed

Insights

Ras mutations can alter binding affinity to PI3K, impacting growth signals. This study quantifies Ras-PI3K binding (KD) using a novel method, revealing mutation-specific effects on this crucial interaction.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Signaling

Background:

  • Ras GTPases are key regulators of cellular signaling pathways.
  • The interaction between Ras and phosphatidylinositol-3-kinase (PI3K) is critical for generating the second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3).
  • Specific Ras mutations are linked to diseases, suggesting altered effector interactions.

Purpose of the Study:

  • To develop and validate a method for quantifying the binding affinity (KD) between active Ras and PI3K.
  • To investigate how Ras mutations at PI3K contact sites affect this binding affinity.
  • To explore the clinical implications of altered Ras-PI3K interactions.

Main Methods:

  • Combined UV spectral deconvolution, High-Performance Liquid Chromatography (HPLC), and microscale thermophoresis.
  • Quantified total Ras concentration and the fraction of active Ras.
  • Measured the binding affinity of active H-Ras and mutants to PI3Kγ Ras-binding domain (RBD) in solution.

Main Results:

  • Developed a precise method for measuring Ras-PI3K binding affinity (KD).
  • Demonstrated that Ras mutations can significantly increase (Q25L), decrease (D38E, Y40C), or have no effect (G13R) on PI3K binding.
  • Showed that quantifying the Ras activation state improves KD measurement accuracy.

Conclusions:

  • Ras mutations can modulate PI3K binding affinity, impacting PIP3 production and downstream signaling.
  • The developed method provides a robust approach to study Ras-effector interactions.
  • Understanding these affinity changes is crucial for deciphering disease mechanisms driven by Ras mutations.
Keywords:
GMPPNGMPPNPGTPHRas GTPaseProtein-nucleotide complexRas small G protein

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