An investigation into the differential phosphorylation patterns of the Ephrin tyrosine kinase receptor, EphA2 in breast and prostate cancers and its interaction with the low molecular weight tyrosine phosphatase, HCPTP

Lake N Paul, Purdue University


The Ephrin tyrosine kinase receptor, EphA2, is overexpressed in breast, prostate, lung and colon cancers. As the grades of cancer progress, the expression level of EphA2 increases, concomitant with a decrease in its tyrosine phosphorylation phenotype. It is hypothesized that EphA2 loses its tyrosine phosphorylation in metastatic cancers because it is not in contact with its ligand, ephrin A1. The restoration of tyrosine phosphorylation on EphA2 can be done by application of either its solublized ligand or by a targeted antibody. As EphA2 contains 17 putative tyrosine phosphorylation sites, the elucidation of which tyrosines are phosphorylated is crucial to understanding the progression of breast and prostate cancers. To determine which tyrosines are phosphorylated under treatment conditions such as pervanadate, EA5 (antibody) and ephrin A1-Fc stimulation, the treated EphA2 was co-immunoprecipitated from breast and prostate cancer cells, and subjected to phospho-peptide mapping analysis. The low molecular weight tyrosine phosphatase, HCPTP, of which there are two isoforms, interacts with the Eph receptors, EphA2, EphB1/B2 and EphA8, though the location of the interaction is conflicting. HCPTP has been shown to dephosphorylate the juxtamembrane residues in EphA8, whereas its primary action is on the SAM domain in the EphB1/B2 system. The exact site of interaction of the phosphatase with EphA2 is unknown. What is known, however, is that HCPTP dephosphorylates EphA2, which in turn promotes tumorigenesis in a non-transformed breast cell line, MCF-10A. The elucidation of the exact site of interaction between the phosphatase and EphA2 was analyzed using Eph derived phospho-peptides, analytical ultracentrifugation and time resolved isotope free mass spectrometry. The results of the phospho-peptide mapping of the co-immunoprecipitated EphA2 from prostate and breast cancer cells revealed a differential phosphorylation pattern in the juxtamembrane region tyrosines 588 and 594, and also on the activation segment loop tyrosine 772. Time resolved mass spectrometry monitoring the dephosphorylation of EphA2 C0 by HCPTP-A and HCPTP-B revealed that HCPTP-A preferentially dephosphorylates Y575, whereas HCPTP-B selectively targets Y588 and Y594. The rates of dephosphorylation at these residues were equivalent. The activation segment tyrosine 772 was dephosphorylated in a similar manner by both isoforms, while the tyrosine 960 from the SAM domain was not dephosphorylated by either HCPTP-A or HCPTP-B.




Stauffacher, Purdue University.

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