B3

Amino Acid Sequence

3D Structure (/structures/AF-P00533-F1-model_v6.cif)

Function

Receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. Known ligands include EGF, TGFA/TGF-alpha, AREG, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF. Ligand binding triggers receptor homo- and/or heterodimerization and autophosphorylation on key cytoplasmic residues. The phosphorylated receptor recruits adapter proteins like GRB2 which in turn activates complex downstream signaling cascades. Activates at least 4 major downstream signaling cascades including the RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC and STATs modules. May also activate the NF-kappa-B signaling cascade. Also directly phosphorylates other proteins like RGS16, activating its GTPase activity and probably coupling the EGF receptor signaling to the G protein-coupled receptor signaling. Also phosphorylates MUC1 and increases its interaction with SRC and CTNNB1/beta-catenin. Positively regulates cell migration via interaction with CCDC88A/GIV which retains EGFR at the cell membrane following ligand stimulation, promoting EGFR signaling which triggers cell migration. Plays a role in enhancing learning and memory performance (By similarity). Plays a role in mammalian pain signaling (long-lasting hypersensitivity) (By similarity). Isoform 2 may act as an antagonist of EGF action. (Microbial infection) Acts as a receptor for hepatitis C virus (HCV) in hepatocytes and facilitates its cell entry. Mediates HCV entry by promoting the formation of the CD81-CLDN1 receptor complexes that are essential for HCV entry and by enhancing membrane fusion of cells expressing HCV envelope glycoproteins.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

To determine whether the reduction in EGFR protein levels induced by B3 resulted from enhanced protein degradation rather than inhibition of protein synthesis, a cycloheximide (CHX) chase assay was performed in HCC827 cells (Fig. 5a). Pretreatment with CHX was used to block de novo protein synthesis, and subsequent exposure to B3 markedly accelerated the decline of EGFR protein levels compared with the CHX-treated control group. These findings indicate that B3 decreases EGFR protein stability and promotes EGFR turnover. We next performed rescue experiments to determine the degradation pathway involved (Fig. 5b). Pretreatment with the proteasome inhibitor MG-132 or the ubiquitin-activating enzyme inhibitor TAK-243 markedly attenuated B3-induced EGFR degradation, suggesting the involvement of the ubiquitin–proteasome pathway. In addition, the autophagy inhibitors BafA1 and chloroquine also rescued EGFR from degradation, indicating that the autophagy pathway may also contribute to B3-mediated EGFR degradation. Given that Hyt induces protein degradation by mimicking misfolded protein-like properties and thereby triggering cellular protein quality control pathways [23, 24], we next examined whether B3 promoted EGFR ubiquitination and Hsp70 association. Co-immunoprecipitation (Co-IP) analysis showed that B3 treatment for 24 h enhanced the interaction between EGFR and Hsp70 and increased the ubiquitination level of EGFR (Fig. 5c). suggesting that Hsp70-mediated ubiquitination is involved in B3-induced EGFR degradation. Taken together, these results support that B3 induces EGFR degradation through an Hsp70-dependent proteasome and autophagy pathway.

Mechanism

The autophagy-lysosomal pathway (ALP) is capable of degrading a broader range of substrates, including longer-lived and larger misfolded proteins and damaged organelles. ALP is classified into three subtypes: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, substrates are directly sequestered and degraded by lysosomes and late endosomes via membrane protrusion and invagination. In CMA, HSPA8/HSC70 recognizes specific substrates containing KFERQ-like sequences; these substrates subsequently bind to LAMP2A on the lysosomal surface and are delivered into lysosomes for degradation. In macroautophagy, substrates are encapsulated through the formation of double-membrane autophagosomes, which subsequently fuse with lysosomes to degrade the substrates.

Description

To determine whether the reduction in EGFR protein levels induced by B3 resulted from enhanced protein degradation rather than inhibition of protein synthesis, a cycloheximide (CHX) chase assay was performed in HCC827 cells (Fig. 5a). Pretreatment with CHX was used to block de novo protein synthesis, and subsequent exposure to B3 markedly accelerated the decline of EGFR protein levels compared with the CHX-treated control group. These findings indicate that B3 decreases EGFR protein stability and promotes EGFR turnover. We next performed rescue experiments to determine the degradation pathway involved (Fig. 5b). Pretreatment with the proteasome inhibitor MG-132 or the ubiquitin-activating enzyme inhibitor TAK-243 markedly attenuated B3-induced EGFR degradation, suggesting the involvement of the ubiquitin–proteasome pathway. In addition, the autophagy inhibitors BafA1 and chloroquine also rescued EGFR from degradation, indicating that the autophagy pathway may also contribute to B3-mediated EGFR degradation. Given that Hyt induces protein degradation by mimicking misfolded protein-like properties and thereby triggering cellular protein quality control pathways [23, 24], we next examined whether B3 promoted EGFR ubiquitination and Hsp70 association. Co-immunoprecipitation (Co-IP) analysis showed that B3 treatment for 24 h enhanced the interaction between EGFR and Hsp70 and increased the ubiquitination level of EGFR (Fig. 5c). suggesting that Hsp70-mediated ubiquitination is involved in B3-induced EGFR degradation. Taken together, these results support that B3 induces EGFR degradation through an Hsp70-dependent proteasome and autophagy pathway.

Description

To further assess the EGFR degradation activity of B3, HCC827 and PC9 cells were treated with increasing concentrations of B3 (0.05–5 μM) for 24 h, followed by Western blot analysis of EGFR protein levels (Fig. 4a). B3 showed DC50 values of 0.61 μM in HCC827 cells and 0.73 μM in PC9 cells.

Description

To further assess the EGFR degradation activity of B3, HCC827 and PC9 cells were treated with increasing concentrations of B3 (0.05–5 μM) for 24 h, followed by Western blot analysis of EGFR protein levels (Fig. 4a). B3 showed DC50 values of 0.61 μM in HCC827 cells and 0.73 μM in PC9 cells

Description

At 5.0 μM, B3 achieved maximal degradation (Dmax) values of 80.4 and 76.3% in HCC827 and PC9 cells

Description

At 5.0 μM, B3 achieved maximal degradation (Dmax) values of 80.4 and 76.3% in HCC827 and PC9 cells

Description

We next evaluated the antiproliferative activity of B3 using a CCK-8 assay. After 72 h of treatment, B3 inhibited the viability of both HCC827 and PC9 cells in a concentration-dependent manner (Fig. 6a), with IC50 values of 171 and 245 nM, respectively.

Description

We next evaluated the antiproliferative activity of B3 using a CCK-8 assay. After 72 h of treatment, B3 inhibited the viability of both HCC827 and PC9 cells in a concentration-dependent manner (Fig. 6a), with IC50 values of 171 and 245 nM, respectively.