Structural Elucidation of the Binding of the Vav‐SH2 Domain to the EphA2 Cytoplasmic Region

Abstract

The Vav guanine nucleotide exchange factors (GEFs) facilitate the GDP/GTP exchange of the Rac/Rho family of small GTPases to modulate cytoskeletal organization, cell morphology and mobility. In endothelial cells, Vav interacts with the EphA2 receptor tyrosine kinase (RTK) with its C‐terminal SH2 domain and plays a crucial role in EphA2‐mediated angiogenesis in cancers. Two closely‐spaced tyrosines in the juxtamembrane segment (JM) of the EphA2 cytoplasmic region, Y588 and Y594, were the postulated Vav‐SH2 recognition sites. Quantitative mass spectrometry data suggests that both JM tyrosines are phosphorylated with high stoichiometry in mammalian cells. However, they exhibit different rates of dephosphorylation by human cytoplasmic protein tyrosine phosphatases (HCPTPs). To characterize the role of each JM phosphotyrosine in Vav‐SH2 binding, isothermal titration calorimetry (ITC) and 15N‐HSQC titration experiments were performed using bacterially expressed Vav1‐SH2 and synthetic JM peptides with different tyrosine phosphorylation patterns. The ITC data indicates Vav1‐SH2 binds preferentially to the doubly phosphorylated and the Y594‐phosphorylated version of JM peptide (JMpYpY and JMYpY), with a KD 10‐fold smaller than the Y588‐phosphorylated version (JMpYY). The 15N‐HSQC spectra suggests all three versions of synthetic JM phosphopeptides bind Vav‐SH2 in similar fashions. The chemical shift perturbation (CSP) analysis of the titration spectra reveals both the primary and secondary phosphotyrosine binding pockets of Vav1‐SH2 are involved in JM phosphopeptide recognition, suggesting each phosphotyrosine has its designated binding pocket. Subsequent 3D NOESY‐HSQC experiments and crystallization trials are undergoing in order to resolve the peptide configuration in the complex of the Vav‐SH2 domain and the JM phosphopeptides.Support or Funding InformationPurdue University Center for Cancer Research