It has been 30 years since the lipid signaling molecule phosphatidylinositol 3,4,5-trisphosphate (PIP3) was first identified¹, and its role as a key regulator of cell growth and survival was recognized in the earliest investigations into this molecule.²

Class IA phosphoinositide 3-kinases (PI3Ks) generate PIP3. The inappropriate activation of this pathway has grave consequences in many human conditions and disease states including many types of cancer, inflammatory processes, and primary immunodeficiencies.³ Class IA PI3Ks are activated downstream of the Ras family of GTPases. Oncogenic Ras is known to promote metabolic reprogramming of tumor cells, prompting them to produce sufficient biomass to support unconstrained proliferation. As such, inhibiting Ras has been called “the holy grail of oncology.”4

But finding strategies to even fully understand Ras-PI3K interactions — let alone finding strategies to inhibit interactions — has been difficult, because these events occur on the surface of the plasma membrane. Biochemists studying membrane-bound events of any kind face challenges due to the unique conformational and temporal constraints that govern such processes in comparison to soluble enzyme processes. But recently, researchers at the University of Victoria in British Columbia, reported success using maleimide-functionalized lipid vesicles to create membrane-resident GTPase HRas (also called transforming protein p21), which allowed them to evaluate its effect on PI3K signaling in lipid kinase assays and through analysis with hydrogen-deuterium exchange mass spectrometry (HDX-MS).³ For the lipid kinase assays, they used the Transcreener® ADP² Fluorescence Intensity (FI) assay.

To do this, they synthesized a lipid film that was used to generate plasma membrane mimic maleimide functionalized vesicles, then conjugated recombinant HRas protein to the vesicles. The material was used for lipid kinase assays by monitoring hydrolysis of ATP, tracking fluorescence intensity (excitation at 590 nm and emission at 620 nm). This allowed them to calculate specific activity using an ATP/ADP standard curve.³ By screening all class IA PI3K isoforms, the researchers found that membrane-resident HRas activates both p110α and p110δ isoforms, but does not activate p110β.5 Further experiments using HDX-MS led them to conclude that membrane-resident HRas was uniquely capable of increasing membrane recruitment of p110α and p110δ, in contrast to soluble HRas.³

The strong activation of PI3Kδ by HRas-coupled vesicles was surprising because previous studies had suggested that HRas does not activate p110δ. The difference in this approach appears to be the use of membrane-localized HRas, suggesting that this system may bear high biological relevance.

The research team is optimistic about the feasibility of this approach, writing, “The use of membrane-coupled Ras in PI3K activity assays could play a key role in rapid screening of these inhibitors for their ability to disrupt PI3K-Ras interactions.” As such, application of the Transcreener Assays could pave the way for identification of new small molecule inhibitors of PI3Ks and Ras by future high throughput screening (HTS) approaches, such as GDP or ADP HTS assays. The possibility of progress toward identifying new inhibitors after 30 years of investigating PIP3 and PI3K pathways is exciting indeed.

– Robyn M. Perrin, PhD

References

[1] Whitman M, Downes CP, Keeler M, Keller T, Cantley L. 1988. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature. 332:644–6.

[2] Traynor-Kaplan AE, Harris AL, Thompson BL, Taylor P, Sklar LA. 1988. An inositol tetrakisphosphate-containing phospholipid in activated neutrophils Nature. 334: 353–356.

[3] Siempelkamp BD, Rathinaswamy MK, Jenkins ML, Burke JE. 2017. Molecular mechanism of activation of class IA phosphoinositide 3-kinases (PI3Ks) by membrane-localized HRas. J Biol Chem. 292(29):12256-12266.

[4] Kimmelman AC. 2015. Metabolic Dependencies in RAS-Driven Cancers. Clin Cancer Res. 21(8):1828-34.

[5] Rodriguez-Viciana P, Sabatier C, McCormic, F. (2004) Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate. Mol. Cell. Biol. 24: 4943–4954.

[6] Gentile DR, Rathinaswamy MK, Jenkins ML, Moss SM, Siempelkamp BD, Renslo AR, Burke JE, Shokat KM. 2017. Ras Binder Induces a Modified Switch-II Pocket in GTP and GDP States. Cell chemical biology. 24(12):1455-66.

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