Fluorescent Biosensor for Measuring Ras Activity in Living Cells

Abstract

The small GTPase Ras is a critical regulator of cell growth and proliferation. Its activity is frequently dysregulated in cancers, prompting decades of work to pharmacologically target Ras. Understanding Ras biology and developing effective Ras therapeutics both require probing Ras activity in its native context, yet tools to measure its activities in cellulo are limited. Here, we developed a ratiometric Ras activity reporter (RasAR) that provides quantitative measurement of Ras activity in living cells with high spatiotemporal resolution. We demonstrated that RasAR can probe live-cell activities of all the primary isoforms of Ras. Given that the functional roles of different isoforms of Ras are intimately linked to their subcellular distribution and regulation, we interrogated the spatiotemporal regulation of Ras utilizing subcellularly targeted RasAR and uncovered the role of Src kinase as an upstream regulator to inhibit HRas. Furthermore, we showed that RasAR enables capture of KRasG12C inhibition dynamics in living cells upon treatment with KRasG12C covalent inhibitors, including ARS1620, Sotorasib, and Adagrasib. We found in living cells a residual Ras activity lingers for hours in the presence of these inhibitors. Together, RasAR represents a powerful molecular tool to enable live-cell interrogation of Ras activity and facilitate the development of Ras inhibitors.

Genetically encodable biosensors for Ras activity

“…More recently, we used our new FRET-based Ras sensor, RasAR, which omits full-length Ras from the sensing unit and enables direct measurements of cellular Ras activity, to probe the effect of Src inhibition on EGF-stimulated HRasWT activity. 34 In COS7 cells coexpressing RasAR and HRasWT, treatment with the Src inhibitors PP2 or SrcI1 yielded enhanced RasAR response compared with the control (i.e., no Src inhibition), suggesting an inhibitory role for Src on Ras signalling. Notably, this effect was only observed when we targeted RasAR to the Golgi membrane and not with the plasma membrane-targeted RasAR.…”

PSL Chemical Biology Symposia Third Edition: A Branch of Science in its Explosive Phase

“…Their FRET-based kinase activity reporter (KAR), enabling an energy transfer upon phosphorylation-induced proximity between two fluorophores, helped decipher protein kinase A (PKA), AMP-dependent protein kinase (AMPK) and other mechanism of action of kinases and phosphatases, [47] cAMP nanodomains in the cAMP/ PKA signaling pathway, [48] and spatiotemporal regulation of phosphoinositides and Akt in the PI3K/Akt/mTORC1 pathway or to quantify Ras activity in the Ras/ERK signaling pathway. [49] Ultrasensitive and super-resolution imaging technologies are also being developed, such as engineered novel ExRai biosensors and Halo-KAR or the FLINC methodology, where the red fluorophore blinks upon phosphorylation of the substrate within the FLINC-KAR by the kinase of interest. [50] Their technology led to the clarification of an ongoing debate about GPCR-mediated ERK signaling.…”

Photoswitchable binders enable temporal dissection of endogenous protein function

“…Various mechanisms have been proposed for late phase signaling from EGFR to ERK 55,56 , however, unequivocal evidence for the role of Ras activity has not been reported. Dynamic signaling of untagged Ras in live cells can be studied by observing Ras activation via biosensors [58][59][60] or chemo-/optogenetic activation of Ras 36,[61][62][63] , but acute loss-of-function experiments were, until recently, unfeasible due to the lack of small-molecule inhibitors of wild-type Ras. The unique ability of psK27.ex17 to inactivate endogenous Ras with precise timing allows us to address if Ras activity is required for late phase ERK activity.…”