In an article recently published by Prentzell et al in Cell, scientists used around 30 different Cell Signaling Technology® (CST®) antibodies to characterize the Ras GTPase-activating protein-binding proteins G3BP1 and G3BP2. These proteins are widely used as markers of stress granules (SGs) - cytoplasmic mRNA-protein complexes that form under adverse environmental conditions and function to promote cell survival by reducing protein synthesis.
As a result of this research, it is now known that G3BPs tether the tuberous sclerosis complex (TSC) to lysosomes and suppress signaling by mechanistic target of rapamycin complex 1 (mTORC1). This finding is significant since mTORC1 hyperactivity has been linked to diseases typified by cellular overgrowth, migration, and neuronal excitability. G3BPs may therefore be important therapeutic targets for cancer and neuronal disorders.
Because all CST antibodies are rigorously validated according to the Hallmarks of Antibody Validation™, the researchers were able to efficiently achieve clear results. These included confirmation of a physical interaction between G3BPs and the TSC complex, and thorough investigation of mTORC1 signaling.
G3BP1 Inhibits mTORC1 in Cells without SGs
Although SGs are known to inhibit mTORC1, the involvement of G3BP1 in this process has, until now, been unclear. To address this, mTORC1 activity was compared between MCF-7 cells treated with arsenite to induce SGs, and cells cultured under conditions unassociated with SG formation.
As expected, arsenite enhanced phosphorylation of the mTORC1 substrate ribosomal protein S6 kinase B1 (RPS6KB1) at T389 (RPS6KB1-pT389). However, arsenite treatment had no effect on RPS6KB1-pT389 levels in G3BP1 knockdown cells, indicating that G3BP1 does not affect mTORC1 activity in cells containing SGs.
Combined with other data, these results verified that mTORC1 inhibition by G3BP1 occurs in the absence of SGs and indicated an additional role for G3BP1 besides that of being a core SG component.
G3BPs Reside at the Lysosomal Surface
A series of studies showed that while G3BP2 can substitute for G3BP1 in SG assembly, it cannot compensate for its loss; G3BP2 does not suppress RPS6KB1-T389 hyperphosphorylation in G3BP1 knockout cells. This finding contributed to the conclusion that G3BPs form a heterocomplex, prompting further investigation to identify the subcellular compartment where these proteins act to inhibit mTORC1.
G3BP1 and G3BP2 were shown to reside in the same fractions as the TSC complex (comprising TSC1, TSC2, and TBC1D7) - namely the lysosomal fractions. Interrogation of these fractions revealed the G3BPs to be localized in close proximity to the protein lysosomal associated membrane protein 1 (LAMP1). However, while LAMP1 (a predominantly luminal protein) remained intact following trypsin protease treatment of lysosomal preparations, the G3BPs and the TSC complex were degraded, indicating the G3BPs to reside at the lysosomal surface with the TSC complex.
Co-immunoprecipitation (co-IP) studies and proximity ligation assays (PLAs) subsequently demonstrated a physical interaction between the G3BPs and the TSC complex. Importantly, this work showed G3BP1 knockout cells to have a similar phenotype to cells exhibiting a TSC2 deficiency, being enlarged and displaying enhanced lysosomal localization of mTOR upon starvation.
G3BP1 Suppresses mTORC1 via the TSC Complex
The interaction between G3BP1 and the TSC complex was explored by analyzing the effect of G3BP1 inhibition on mTORC1 activity in the presence or absence of TSC2. While G3BP1 inhibition induced RPS6KB1-pT389 in control cells, a similar outcome was not seen in TSC2 knockouts (where the TSC complex is lacking). These results suggest that G3BP1 and the TSC complex act in the same pathway to suppress mTORC1, with the TSC complex being dependent on G3BP1 as its lysosomal tether.
Targeting G3BP1 for Therapeutic Intervention
G3BP1 has significant therapeutic potential for diseases characterized by mTORC1 hyperactivity. In the publication, it was noted that relapse-free survival (RFS) of individuals with breast cancer is shorter where G3BP1 mRNA or protein levels fall below the median, a finding that aligns with the shorter RFS in patients with low TSC1 or TSC2. Exploiting G3BP1 may therefore increase patient survival rates, for example by preventing metastasis or slowing tumor growth.
Additionally, the publication reported that inhibition of zebrafish G3bp1 has been shown to enhance mTORC1 activity, as evidenced by phosphorylation of ribosomal protein s6 at serine 235/236 (Rps6-pS235/236). Where such inhibition takes place in vivo, it phenocopies the mTORC1-dependent effects of Tsc2 loss on brain function, emphasizing the critical role of G3bp1 within nervous system development and activity. This highlights the possible utility of G3BPs in treating neurological disorders.
To learn more about how CST antibodies were used to study G3BPs and reveal how these essential proteins might be targeted to treat cancer and neuronal disorders, read the full article here.