EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling

Singh, Deo R.; Ahmed, Fozia; King, Christopher R.; Gupta, Nisha; Salotto, Matt; Pasquale, Elena B.; Hristova, Kalina

doi:10.1074/jbc.m115.676866

Cited by 60 publications

(102 citation statements)

References 42 publications

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“…The production of the pcDNA 3.1(+) EphA2-15aa-mTurq and pcDNA3.1(+) EphA2-15aa-eYFP constructs has been described previously (39). These constructs encode full-length EphA2, followed by a flexible linker consisting of 5 GGS repeats, and fluorescent proteins (mTurqouise and eYFP, a FRET pair) at the C-terminus.…”

Section: Methodsmentioning

confidence: 99%

“…While EphA2 was believed to be monomeric when not bound to ephrin ligands, we have recently demonstrated that this receptor can form dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding (39). Furthermore, we have shown that mutation to Arg of three hydrophobic amino acids in the extracellular cysteine-rich region (L223, L254, and V255) destabilizes the EphA2 dimer.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, we have shown that mutation to Arg of three hydrophobic amino acids in the extracellular cysteine-rich region (L223, L254, and V255) destabilizes the EphA2 dimer. The dimerization-deficient EphA2 mutant shows enhanced ability to promote cell migration concomitant with increased phosphorylation on Ser897 and decreased tyrosine phosphorylation as compared to EphA2 wild-type (39). These findings suggest that the EphA2 monomer favors pro-tumorigenic signaling.…”

Section: Introductionmentioning

confidence: 99%

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A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers

Singh

Pasquale

Hristova

2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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BACKGROUND The EphA2 receptor tyrosine kinase is known to promote cancer cell malignancy in the absence of activation by ephrin ligands. This behavior depends on high EphA2 phosphorylation on Ser897 and low tyrosine phosphorylation, resulting in increased cell migration and invasiveness. We have previously shown that EphA2 forms dimers in the absence of ephrin ligand binding, and that dimerization of unliganded EphA2 can decrease EphA2 Ser897 phosphorylation. We have also identified a small peptide called YSA, which binds EphA2 and competes with the naturally occurring ephrin ligands. METHODS Here, we investigate the effect of YSA on EphA2 dimer stability and EphA2 function using quantitative FRET techniques, Western blotting, and cell motility assays. RESULTS We find that the YSA peptide stabilizes the EphA2 dimer, increases EphA2 Tyr phosphorylation, and decreases both Ser897 phosphorylation and cell migration. CONCLUSIONS The experiments demonstrate that the small peptide ligand YSA reduces EphA2 Ser897 pro-tumorigenic signaling by stabilizing the EphA2 dimer. GENERAL SIGNIFICANCE This work is a proof-of-principle demonstration that EphA2 homointeractions in the plasma membrane can be pharmacologically modulated to decrease the pro-tumorigenic signaling of the receptor.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers

Singh

Pasquale

Hristova

2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…Additionally, the SAM domain was reported to be required for Ser-897 phosphorylation and its noncanonical function, suggesting that the SAM domain stabilizes the kinase approach or inhibits the phosphatase approach to Ser-897. 49,50) Moreover, EphA2 prefers the dimer formation in the absence of ephrin-A1. This dimerization suppresses the phosphorylation of Ser-897 and attenuates the function of Ser-897 in comparison with the EphA2 monomer, indicating that the substrate-kinase reaction for EphA2 on Ser-897 is more stable in the EphA2 mononer than in the dimer.…”

Section: Epha2 Noncanonical Signaling Transductionmentioning

confidence: 99%

Emerging and Diverse Functions of the EphA2 Noncanonical Pathway in Cancer Progression

Zhou

Sakurai

2017

Biological & Pharmaceutical Bulletin

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Erythropoietin-producing hepatocellular receptor A2 (EphA2) receptor tyrosine kinase controls multiple physiological processes to maintain homeostasis in normal cells. In many types of solid tumors, it has been reported that EphA2 is overexpressed and plays a critical role in oncogenic signaling. However, in recent years, the opposing functions of EphA2 have been explained by the canonical and noncanonical signaling pathways. Ligand-and tyrosine kinase-dependent EphA2 activation (the canonical pathway) inhibits cancer cell proliferation and motility. In contrast, ligand-and tyrosine kinase-independent EphA2 signaling (the noncanonical pathway) promotes tumor survival and metastasis and controls acquired drug resistance and maintenance of cancer stem cell-like properties. Evidence has accumulated showing that the EphA2 noncanonical pathway is mainly regulated by inflammatory cytokines and growth factors via phosphorylation at Ser-897 in the intracellular C-tail region via some serine/threonine kinases, including p90 ribosomal S6 kinase. In this review, we focus on the regulation of Ser-897 phosphorylation and its functional importance in tumor malignancy and discuss future therapeutic targeting.

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“…As a result of the osmotic stress, cells flatten their caveolae (60–80 nm cup-shaped invaginations) and “un-wrinkle” their membranes (29). Hypoosmotic swelling results in a simple vesicle-like membrane topology (18,30,31), allowing for the determination of two-dimensional receptor concentrations in the membrane (18). Here we use such live cells under reversible osmotic stress to characterize the interactions between the EC-TM VEGFR2 constructs in FRET experiments.…”

Section: Resultsmentioning

confidence: 99%

Cooperative interactions between VEGFR2 extracellular Ig-like subdomains ensure VEGFR2 dimerization

King

Wirth

Workman

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Prior studies have suggested that the interactions occurring between VEGFR2 extracellular domains in the absence of ligand are complex. Here we seek novel insights into these interactions, and into the role of the different Ig-like domains (D1 through D7) in VEGFR2 dimerization. Methods We study the dimerization of a single amino acid mutant and of three deletion mutants in the plasma membrane using two photon microscopy and fully quantified spectral imaging. Results We demonstrate that a set of cooperative interactions between the different Ig-like domains ensure that VEGFR2 dimerizes with a specific affinity instead of forming oligomers. Conclusions The contributions of subunits D7 and D4 seem to be the most critical, as they appear essential for strong lateral interactions and for the formation of dimers, respectively. General Significance This study provides new insights into the mechanism of VEGFR2 dimerization and activation.

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EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling

Cited by 60 publications

References 42 publications

A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers

A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers

Emerging and Diverse Functions of the EphA2 Noncanonical Pathway in Cancer Progression

Cooperative interactions between VEGFR2 extracellular Ig-like subdomains ensure VEGFR2 dimerization

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