Interaction of EphA2 with human cytoplasmic protein tyrosine phosphatase (HCPTP) variants and phosphorylation status of EphA2 in cancer cells by quantitative selected reaction monitoring mass spectrometry (SRM MS)

Deepa Balasubramaniam, Purdue University

Abstract

The EphA2 receptor tyrosine kinase and the human cytoplasmic protein tyrosine phosphatase (HCPTP) are overexpressed in a number of epithelial cancers. Overexpressed EphA2 in these cancers shows a significant decrease in phosphotyrosine content which results in suppression of receptor signaling and endocytosis and an increase in metastatic potential. The decreased phosphotyrosine content of EphA2 has been associated with decreased contact with its ligand, ephrin A1 and dephosphorylation by HCPTP. Potential specificity of the two HCPTP variants for tyrosines on EphA2 has not been investigated. A mass spectrometry assay has been used to measure relative rates of dephosphorylation for the two HCPTP variants at phosphotyrosine sites associated with control of the EphA2 kinase activity or interaction with downstream targets. In order to quantitate our dephosphorylation studies a mass spectrometry based method to determine the stoichiometry of protein post translational modification (PTM) was developed. Quantification of protein and PTM abundance in biological samples is an important component of proteomic studies. Label-free methods for quantification using mass spectrometry (MS) are attractive because they are simple to implement and applicable to any experimental system. Our studies demonstrate that PTM stoichiometry can be accurately measured using label-free quantification and selected reaction monitoring (SRM). Use of selected reaction monitoring is advantageous with complex biological samples and our results show that this approach can be used to quantify multiple PTMs independently on a single peptide. The results of our specificity studies suggest that although both variants of HCPTP can dephosphorylate the EphA2 receptor, the rate and specificity of dephosphorylation for specific tyrosines are different for HCPTP-A and -B. The SAM domain tyrosine Y960 which has been implicated in downstream PI3K signaling is dephosphorylated exclusively by HCPTP-B. The activation loop tyrosine (Y772) which directly controls kinase activity is dephosphorylated about 6 times faster by HCPTP-A. In contrast, the juxtamembrane tyrosines (Y575, Y588 and Y594) which are implicated in both control of kinase activity and downstream signaling are dephosphorylated by both variants with similar rates. This difference in preference of dephosphorylation sites on EphA2 not only illuminates the different roles of the two variants of the phosphatase in EphA2 signaling, but also explains why both HCPTP variants are highly conserved in most mammals. The quantitative SRM-MS method was then applied to transformed prostate and breast cell lines to study the change in phosphorylation on specific tyrosines upon treatment with reagents designed to reestablish normal EphA2 signaling, an EphA2 antibody (EA5), ephrinA1 Fc (B61) and pervanadate. Results from these experiments show that there is a statistically significant increase in phosphorylation at pY772, pY575 and ppY588Y594 for all three treatments tested. These results confirm that the activation loop and juxtamembrane tyrosines that are predicted to be involved in regulating kinase activity and play an important role in downstream signaling are downregulated in a transformed cell line. The observed increase in phosphorylation indicates this can be reversed with all of the above treatments. Previous studies have identified a potential secondary binding site for peptide substrates on the HCPTP variants. 1-naphthylacetic acid (1NA) has been shown to bind this secondary site on HCPTP-A and -B by crystallography and NMR, but it does not appear affect activity when assayed with pNPP as substrate. To study the effect of binding of 1NA on the natural substrate EphA2 C0, we applied the SRM MS method to study the dephosphorylation kinetics of EphA2 tyrosines by HCPTP-A. Results of these studies suggest that there is in an increase in the overall rate of dephosphorylation of EphA2 tyrosines by HCPTP-A in the presence of 1NA.

Degree

Ph.D.

Advisors

Stauffacher, Purdue University.

Subject Area

Biochemistry|Biophysics|Oncology

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