EGFR forms ligand-independent oligomers that are distinct from the active state.
Ontology highlight
ABSTRACT:
SUBMITTER: Byrne PO
PROVIDER: S-EPMC7504936 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Selection of activating EGFR mutations from randomly mutated EGFR libraries
| PRJEB49811 | ENA
Project description:The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with important roles in many cellular processes as well as in cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. How these dimers relate to higher-order EGFR oligomers seen in cell membranes, however, remains unclear. Here, we used single-particle tracking (SPT) and Förster resonance energy transfer imaging to examine how each domain of EGFR contributes to receptor oligomerization and the rate of receptor diffusion in the cell membrane. Although the extracellular region of EGFR is sufficient to drive receptor dimerization, we find that the EGF-induced EGFR slowdown seen by SPT requires higher-order oligomerization-mediated in part by the intracellular tyrosine kinase domain when it adopts an active conformation. Our data thus provide important insight into the interactions required for higher-order EGFR assemblies involved in EGF signaling.
| S-EPMC10835193 | biostudies-literature
Project description:The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.
| S-EPMC4751368 | biostudies-literature
Project description:Identification of novel targets for triple-negative breast cancer (TNBC) is an urgent task as targeted therapies have increased the lifespans of Oestrogen Receptor +/ Progesterone Receptor + and HER2+ cancer patients. Methods: genes involved in protein processing in the endoplasmic reticulum, which have been reported to be key players in cancer, were used in loss-of-function screening to evaluate the oncogenic roles of these genes to identify candidate target genes in TNBC. In vitro and in vivo function assays as well as clinical prognostic analysis were used to study the oncogenic role of the gene. Molecular and cell based assays were further employed to investigate the mechanisms. Results: B Cell Receptor Associated Protein 31 (BCAP31), the expression of which is correlated with early recurrence and poor survival among patients, was identified an oncogene in our assay. In vitro studies further suggested that BCAP31 acts as a key oncogene by promoting TNBC development. We also showed that BCAP31 interacts with epidermal growth factor receptor (EGFR) and serves as an inhibitor of ligand-independent EGFR recycling, sustaining EGFR autophosphorylation and activation of downstream signalling. Conclusion: These findings reveal the functional role of BCAP31, an ER-related protein, in EGFR dysregulation and TNBC development.
| S-EPMC6771250 | biostudies-other
Project description:We describe here an important function of the novel calmodulin kinase I isoform, pregnancy-upregulated nonubiquitous calmodulin kinase (Pnck). Pnck (also known as CaM kinase Ibeta(2)) was previously shown to be differentially overexpressed in a subset of human primary breast cancers, compared with benign mammary epithelial tissue. In addition, during late pregnancy, Pnck mRNA was shown to be strongly upregulated in epithelial cells of the mouse mammary gland exhibiting decreased proliferation and terminal differentiation. Pnck mRNA is also significantly upregulated in confluent and serum-starved cells, compared with actively growing proliferating cells (Gardner HP, Seung HI, Reynolds C, Chodosh LA. Cancer Res 60: 5571-5577, 2000). Despite these suggestive data, the true physiological role(s) of, or the signaling mechanism(s) regulated by Pnck, remain unknown. We now report that epidermal growth factor receptor (EGFR) levels are significantly downregulated in a ligand-independent manner in human embryonic kidney-293 (HEK-293) cells overexpressing Pnck. MAP kinase activation was strongly inhibited by EGFR downregulation in the Pnck-overexpressing cells. The EGFR downregulation was not the result of reduced transcription of the EGFR gene but from protea-lysosomal degradation of EGFR protein. Knockdown of endogenous Pnck mRNA levels by small interfering RNA transfection in human breast cancer cells resulted in upregulation of unliganded EGFR, consistent with the effects observed in the overexpression model of Pnck-mediated ligand-independent EGFR downregulation. Pnck thus emerges as a new component of the poorly understood mechanism of ligand-independent EGFR degradation, and it may represent an attractive therapeutic target in EGFR-regulated oncogenesis.
| S-EPMC2518423 | biostudies-literature
Project description:In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in ?-sheet rich oligomers with increased ?-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.
| S-EPMC5970100 | biostudies-literature
Project description:The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. We previously showed that the pivotal effector of this pathway, YAP, is amplified in tumors and promotes epithelial-to-mesenchymal transition (EMT) and malignant transformation. Here, we report that overexpression of TAZ, a paralog of YAP, in human mammary epithelial cells promotes EMT and, in particular, some invasive structures in 3D cultures. TAZ also leads to cell migration and anchorage-independent growth in soft agar. Furthermore, we identified amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand, as a target of TAZ. We show that AREG functions in a non-cell-autonomous manner to mediate EGF-independent growth and malignant behavior of mammary epithelial cells. In addition, ablation of TEAD binding completely abolishes the TAZ-induced phenotype. Last, analysis of breast cancer patient samples reveals a positive correlation between TAZ and AREG in vivo. In summary, TAZ-dependent secretion of AREG indicates that activation of the EGFR signaling is an important non-cell-autonomous effector of the Hippo pathway, and TAZ as well as its targets may play significant roles in breast tumorigenesis and metastasis.
| S-EPMC3419062 | biostudies-literature
Project description:PurposeThe aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling.Materials and methodsMET+ and EGFR+ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control ± HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining.ResultsHeat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling.ConclusionsHeat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.
| S-EPMC6175538 | biostudies-literature
Project description:RhoU is an atypical member of the Rho family of small G-proteins, which has N- and C-terminal extensions compared to the classic Rho GTPases RhoA, Rac1 and Cdc42, and associates with membranes through C-terminal palmitoylation rather than prenylation. RhoU mRNA expression is upregulated in prostate cancer and is considered a marker for disease progression. Here, we show that RhoU overexpression in prostate cancer cells increases cell migration and invasion. To identify RhoU targets that contribute to its function, we found that RhoU homodimerizes in cells. We map the region involved in this interaction to the C-terminal extension and show that C-terminal palmitoylation is required for self-association. Expression of the isolated C-terminal extension reduces RhoU-induced activation of p21-activated kinases (PAKs), which are known downstream targets for RhoU, and induces cell morphological changes consistent with inhibiting RhoU function. Our results show for the first time that the activity of a Rho family member is stimulated by self-association, and this is important for its activity.
| S-EPMC10917059 | biostudies-literature
Project description:We have recently described a novel role for pregnancy-upregulated non-ubiquitous calmodulin kinase (Pnck) in the induction of ligand-independent epidermal growth factor receptor (EGFR) degradation (Deb TB, Coticchia CM, Barndt R, Zuo H, Dickson RB, and Johnson MD. Am J Physiol Cell Physiol 295: C365-C377, 2008). In the current communication, we explore the probable mechanism by which Pnck induces ligand-independent EGFR degradation. Pnck-induced EGFR degradation is calcium/calmodulin independent and is regulated by cell density, with the highest EGFR degradation observed at low cell density. Pnck is a novel heat shock protein 90 (Hsp90) client protein that can be co-immunoprecipitated with Hsp90. Treatment of Pnck-overexpressing cells with the pharmacologic Hsp90 inhibitor geldanamycin results in enhanced EGFR degradation, and destruction of Pnck. In cells in which Pnck is inducing EGFR degradation, we observed that Hsp90 exhibits reduced electrophoretic mobility, and through mass spectrometric analysis of immunopurified Hsp90 protein we demonstrated enhanced phosphorylation at threonine 89 and 616 (in both Hsp90-? and -?) and serine 391 (in Hsp90-?). Kinase-active Pnck protein is degraded by the proteasome, concurrent with EGFR degradation. A Pnck mutant (T171A) protein with suppressed kinase activity induced EGFR degradation to essentially the same level as wild-type (WT) Pnck, suggesting that Pnck kinase activity is not required for the induction of EGFR degradation. Although EGFR is degraded, overexpression of WT Pnck paradoxically promoted cellular proliferation, whereas cells expressing mutant Pnck (T171A) were growth inhibited. WT Pnck promoted S to G(2) transition, but cells expressing the mutant exhibited higher residency time in S phase. Basal MAP kinase activity was inhibited by WT Pnck but not by mutant T171A Pnck protein. Cyclin-dependent kinase (Cdk) inhibitor p21/Cip-1/Waf-1 was transcriptionally suppressed downstream to MAP kinase inhibition by WT Pnck, but not the mutant protein. Collectively, these data suggest that 1) Pnck induces ligand-independent EGFR degradation most likely through perturbation of Hsp90 chaperone activity due to Hsp90 phosphorylation, 2) EGFR degradation is coupled to proteasomal degradation of Pnck, and 3) modulation of basal MAP kinase activity, p21/Cip-1/Waf-1 expression, and cellular growth by Pnck is independent of Pnck-induced ligand-independent EGFR degradation.
| S-EPMC3093937 | biostudies-literature