A recent study published in Cell Research revealed that lysosome-localized epidermal growth factor receptor (EGFR) directly binds to and activates Rheb, a key activator of mTORC1, in a kinase activity-independent manner. Mechanistically, EGFR utilizes its Glu804 residue, which likely functions as a catalytic “glutamic finger”, to exert a novel role as a guanine nucleotide exchange factor for Rheb.

The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved multi-subunit kinase complex centered around the serine/threonine kinase, mTOR.1 It functions as a critical regulatory hub that integrates diverse upstream cues, including nutrient availability, growth factor signaling, cellular energy status, and environmental stress, to govern cellular growth and proliferation events.2 Through its kinase activity, mTORC1 phosphorylates a plethora of key downstream effectors — including S6K1, 4EBP1, and ULK1, thereby promoting anabolic processes and suppressing catabolic pathways to coordinate cellular growth.3 Over the past two decades, it has been established that mTORC1 activation is regulated mainly by two major signaling pathways (Fig. 1). The first pathway involves growth factor signaling, which regulates the activity of Rheb via the PI3K/AKT axis to govern mTORC1 kinase activity. The second pathway engages with amino acid signaling, which modulates the activity of Rag GTPases (RagA/B and RagC/D) to promote the recruitment of mTORC1 to the lysosomal surface.4,5 Both Rheb and Rag are small GTPases whose activity largely depends on their nucleotide-binding status. The nucleotide-binding states are dynamically regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs).4,5

Fig. 1: Schematic representation of lysosomal EGFR functioning as a GEF for Rheb to promote the activation of the mTORC1 signaling pathway.
figure 1

Amino acid signals are sensed by intracellular amino acid sensors and relayed through the GATOR2-GATOR1-Ragulator signaling axis to regulate the nucleotide-binding states of Rag GTPases (RagA/B and RagC/D), thereby mediating the recruitment of mTORC1 to the lysosomal membrane. In this cascade, GATOR1 acts as a GAP for RagA/B, while Ragulator functions as a GEF for RagA/B. Concurrently, growth factor signals regulate the nucleotide-binding status of Rheb via two distinct mechanisms: (1) through canonical EGFR kinase activity and downstream PI3K/AKT/TSC2 signaling, where TSC2 serves as a GAP for Rheb, and this GAP activity can be inhibited by AKT-mediated phosphorylation, and (2) through lysosome-localized EGFR, which directly binds to Rheb and acts as its GEF to facilitate Rheb activation independently of EGFR kinase activity. Activated Rags and Rheb at the lysosome surface promote spatiotemporal mTORC1 kinase activation, leading to phosphorylation of downstream effectors and ultimately driving cell growth and proliferation. Figure created with BioRender.com.

Full size image

Notably, the growth factor signaling cascade activates the PI3K/AKT pathway, which phosphorylates and inhibits TSC2 — a GAP for Rheb — thereby promoting the GTP-bound, active state of Rheb and the subsequent activation of mTORC1.1,2 However, despite extensive research over several decades, a bona fide GEF that activates Rheb has remained elusive. On the other hand, Rag GTPases function as obligate heterodimers anchored to the lysosomal membrane. The active heterodimeric conformation consists of GTP-bound RagA/B and GDP-bound RagC/D, whereas the inactive conformation features GDP-bound RagA/B and GTP-bound RagC/D.4 Importantly, Ragulator, a pentameric complex composed of p18, p14, C7orf59, HBXIP, and MP1, acts as a GEF for RagA/B, promoting their GTP-bound state. Conversely, the GATOR1 complex, consisting of DEPDC5, NPRL2, and NPRL3, functions as a GAP that facilitates the hydrolysis of GTP on RagA/B, thus promoting the inactive, GDP-bound state.5 This dynamic cycling between GTP- and GDP-bound states allows Rag GTPases to sense changes in amino acid availability and to regulate the lysosomal localization of mTORC1 in a timely manner.

Epidermal growth factor receptor (EGFR) is a membrane-anchored tyrosine kinase. Aberrant EGFR activity, often resulting from gene amplification or gain-of-function mutations, is commonly observed in tumorigenesis, particularly in non-small cell lung cancer (NSCLC).6,7 These oncogenic mutations confer constitutive, ligand-independent activation of EGFR and are frequently accompanied by abnormal intracellular trafficking, with mutant EGFR localizing to both early/recycling endosomes and late endosomal/lysosomal compartments.7 Clinical trials have demonstrated that second-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as afatinib, significantly prolong progression-free survival (PFS) in patients with EGFR-mutant NSCLC, compared to first-generation inhibitors like erlotinib, but the underlying mechanisms remain incompletely understood.8 EGFR appears to act as an upstream regulator of Rheb through multiple signaling routes. Upon binding to its ligand EGF, EGFR undergoes activation and autophosphorylation, subsequently initiating the downstream PI3K/AKT/TSC2 signaling cascade, which ultimately leads to mTORC1 activation to favor tumorigenesis.4 Now the Kang group identified that lysosome-localized EGFR can activate Rheb independently of its well-characterized kinase function.9 Further investigation from the Kang group revealed that the tyrosine kinase ___domain (TKD) of EGFR is responsible for binding to Rheb to mediate downstream activation of mTORC1.9 Interestingly, this interaction is selectively disrupted by afatinib, but not by erlotinib, thereby providing a mechanistic insight into the superior clinical efficacy of afatinib.9 These findings point to a previously unrecognized function of EGFR in modulating mTORC1 activity through direct, kinase-independent binding to Rheb, and suggest that targeting both kinase and non-kinase functions of EGFR may represent a more effective therapeutic strategy to combat cancers.

Structural prediction using AlphaFold2 suggested that EGFR-TKD interacts with Rheb at the nucleotide-binding pocket, with the side chain of Glu804 identified as a potential “glutamic finger” crucial for the putative GEF activity of EGFR.9 In keeping with this notion, this residue is highly conserved across multiple vertebrate species. Further experimental validation using EGFR-E804K knock-in cells revealed a significant reduction in the binding affinity between EGFR and Rheb, accompanied by a marked decrease in the levels of GTP-bound Rheb. Consequently, mTORC1 activity was substantially diminished in EGFR-E804K knock-in cells.9 In addition, cellular assays demonstrated that the proliferative capacity of EGFR-E804K knock-in cells was significantly impaired.9 Taken together, these findings indicate that the Rheb-GEF activity of EGFR is likely essential for mTORC1 activation and represents a critical determinant of cell growth.

Given the considerable toxicity associated with afatinib in clinical applications,8 the authors rationally designed a novel small-molecule compound that targets EGFR through simultaneous inhibition of both its kinase activity and the newly defined Rheb-GEF activity. This compound was named BIEGi-1 (binary EGFR inhibitor). Compared to afatinib, BIEGi-1 suppresses the GEF activity of EGFR toward Rheb more effectively, and correspondingly exhibits enhanced anti-proliferative effects on cells harboring EGFR mutations.9 These findings underscore the therapeutic potential of concurrently targeting both the kinase and the Rheb-GEF activities of EGFR as a promising strategy for inhibiting the growth of cancer cells with EGFR mutation.

This study also raises several important questions that warrant further in-depth investigation. For instance, a previous independent study identified ATP6AP1 as a putative GEF for Rheb in response to insulin stimulation.10 Hence, it remains unclear whether other Rheb-GEFs exist that respond to oxidative stress or other cellular environmental cues to regulate mTORC1 activity. If so, do these Rheb GEFs function in a redundant or complementary manner? Furthermore, since EGFR lacks a classical GEF ___domain, the precise mechanism by which it catalytically modulates Rheb’s nucleotide-binding state remains to be further elucidated. To this end, high-resolution structural studies, such as X-ray crystallography or cryo-electron microscopy of the EGFR–Rheb complex, will be insightful to uncover the detailed molecular mechanism of this interaction. Additionally, the in vivo efficacy and potential toxicity of BIEGi-1 require thorough evaluation before being considered for clinical applications. Addressing these questions will not only deepen our understanding of EGFR-mediated regulation of Rheb and mTORC1 signaling, but may also offer novel therapeutic strategies for targeting EGFR-driven cancers.