Binding of active Ras and its mutants to the Ras binding domain of PI-3-kinase: A quantitative approach to K_D measurements.

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 K_D for binding. The approach measures the total Ras concentration, the fractio

SUBMITTER: Fleming IR 

PROVIDER: S-EPMC9884175 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature