Specificity of HCPTP variants toward EphA2 tyrosines by quantitative selected reaction monitoring

Abstract

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. We have used a mass spectrometry assay 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. Our results suggest that although both variants dephosphorylate the EphA2 receptor, the rate and specificity of dephosphorylation for specific tyrosines are different for HCPTP-A and HCPTP-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 six 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 for 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.

Figure 1

Comparison of global EphA2 C0 dephosphorylation catalyzed by HCPTP-A and HCPTP-B. Aliquots drawn at various time points were resolved on 12% SDS PAGE and either stained with Coomassie blue or probed with an anti-phosphotyrosine antibody (pY20). (A) Dephosphorylation of EphA2 C0 by HCPTP-A. (B) Dephosphorylation of EphA2 C0 by HCPTP-B. The increase in mobility of EphA2 C0 upon dephosphorylation is indicated by arrows.

Figure 2

Dephosphorylation of EphA2 C0 by HCPTP-A. Samples drawn at various time points were subjected to SRM-MS analysis. The percentage phosphorylation for the sample at 0 min was adjusted to 100%, and the percent phosphorylation over time is plotted relative to 0 min. (A) A comparison of the dephosphorylation profiles for pY772, ppY588Y594, pY575 and pY960. (B) A comparison of the dephosphorylation profiles for the three phosphorylated forms of the peptide containing both pY588 and pY594. All experiments were repeated at least three times and the mean and SD from the mean are shown.

Figure 3

Dephosphorylation of EphA2 C0 by HCPTP-B. Samples drawn at various timepoints were subjected to SRM-MS analysis. The percentage phosphorylation for the sample at 0 min was adjusted to 100%, and the percent phosphorylation over time is plotted relative to 0 min. (A) A comparison of the dephosphorylation profiles for pY772, ppY588Y594, pY575, and pY960. (B) A comparison of the dephosphorylation profiles for the three phosphorylated forms of the peptide containing both pY588 and pY594. All experiments were repeated at least three times and the mean and SD from the mean are shown.

Figure 4

pY588Y594 builds up as a result of dephosphorylation at Y594 on the doubly phosphorylated peptide ppY588Y594 as the reaction proceeds. 10 μM EphA2 C0 was treated with 10 μM HCPTP-A (C0 + A 10:10) or with 1 μM HCPTP-A (C0 + A 10:1) and the SRM-MS data for pY588Y594 was analyzed at various timepoints. The mean and standard deviation of three independent replicates are plotted.

Figure 5

Specificity of HCPTP variants towards EphA2 tyrosines with linked pathways and functions. The juxtamembrane region is shown as an unstructured loop with three phosphorylated tyrosines (Y575, Y588, and Y594) represented by purple circles. The juxtamembrane tyrosines play a role in activation and inhibition of kinase activity, interact with Vav GEFs, and participate in other SH2 mediated reactions. The rate of dephosphorylation of the juxtamembrane by HCPTP variants is comparable. The kinase domain is shown in orange with the activation loop tyrosine (Y772) marked as a purple circle. Y772 is the preferred site for dephosphorylation by HCPTP-A. The SAM domain of EphA2 (red) has been shown to interact with the SAM domain of Ship2 and regulate EphA2 endocytosis through PI3K dependent Rac1 activation. The SAM domain tyrosine, Y960 is a target for dephosphorylation only by HCPTP-B.