Home Blog of Signal Transduction Epigenetics MicroRNA Binding of active Ras and its mutants to the Ras binding domain of PI-3-kinase: A quantitative approach to KD measurements

Category

Archives

Popular Post

ESKM, a novel therapeutic agent for sensitive and resistant PH+ leukemias

The mechanism of resistance to JAK2 inhibitor in myeloproliferative neoplasms patients

Effects of PARP inhibitors in BRCA gene-mutated ovarian cancer

DASATINIB; An inhibitor of Receptor Tyrosine Kinase

GP130/JAK/STAT3 signaling pathway induces multiple myeloma

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

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.

 

Comments:

That's an impressive exploration of Ras GTPases and their interaction with PI3K! The intricate relationship between these molecules and their impact on cellular signaling underscores the complexity of biological systems.

Your hypothesis regarding Ras mutations affecting PI3K binding affinity is quite intriguing, especially considering the clinical implications tied to disease-linked Ras mutations at PI3K contact positions. The developed approach utilizing UV spectral deconvolution, HPLC, and microscale thermophoresis to quantify the KD for binding seems comprehensive and promising for testing this hypothesis.

The ability to measure the total Ras concentration, fraction of active Ras, and the affinity of active Ras binding to PI3K's Ras binding domain in solution provides a detailed understanding of these interactions. The findings revealing how specific mutations in H-Ras (such as Q25L, D38E, Y40C, and G13R) can either increase, decrease, or have no effect on PI3K binding affinity underscore the nuanced impact of mutations on cellular signaling pathways.

These discoveries potentially pave the way for understanding how alterations in Ras affinity can modify PI3K regulation and subsequently influence PIP3 growth signals, shedding light on potential avenues for therapeutic intervention or targeted treatments for diseases associated with Ras mutations affecting PI3K interactions.

Related Products

Cat.No. Product Name Information
S3084 Guanosine 5'-triphosphate trisodium salt

Guanosine 5'-triphosphate (5'-GTP, Guanosine triphosphate, H4GTP) trisodium salt is an enhancer of myogenic cell differentiation in a murine cells. Guanosine 5'-triphosphate trisodium salt up-regulates miRNA (specifically miR133a and miR133b) and myogenic regulator factor and induces human myogenic precursor cells to release exosomes stuffed with guanosine based molecules (mainly guanosine) in the extracellular milieu.

Related Targets

MicroRNA