Biochemical and NMR characterization of the interactions of Vav2–SH2 domain with lipids and the EphA2 juxtamembrane region on membrane

Abstract

Vav2 is a ubiquitous guanine nucleotide exchange factor (GEF) for Rho family GTPases that is involved in regulating a wide range of biological processes. It interacts with several tyrosine-phosphorylated cell surface receptors, including the Eph family receptors, through its SH2 domain. The interaction of Vav2 with EphA2 is crucial for EphA2-mediated tumor angiogenesis. Here we show that Vav2-SH2 domain is a lipid-binding module that can recognize PI(4,5)P2 and PI(3,4,5)P3 lipids weakly but specifically. The specific lipid-binding site in Vav2-SH2 domain was identified by NMR chemical shift perturbation experiments using the head groups of PI(4,5)P2 and PI(3,4,5)P3, both of which bind to Vav2-SH2 with millimolar binding affinities. In addition, the interaction between Vav2-SH2 and the phosphorylated juxtamembrane region (JM) of EphA2 (Y594 phosphorylated) was investigated using NMR techniques. Furthermore, by using a nickel-lipid containing peptide-based nanodiscs system, we studied the binding of Vav2-SH2 to the phosphorylated JM region of EphA2 on lipid membrane and uncovered a role of membrane environment in modulating this protein-protein recognition.

Novel Roles of SH2 and SH3 Domains in Lipid Binding

“…One of the most recent studies on SH2 domain–lipid interactions focused on Vav2 [ 59 ], a ubiquitous guanine nucleotide exchange factor for Rho family GTPases that is involved in the regulation of a wide variety of biological processes [ 60 , 61 ]. Via its SH2 domain, Vav2 interacts with many different tyrosine-phosphorylated cell-surface receptors [ 62 ].…”

“…Via its SH2 domain, Vav2 interacts with many different tyrosine-phosphorylated cell-surface receptors [ 62 ]. Ge et al have reported that the Vav2 SH2 domain can specifically recognize phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate but binds them weakly [ 59 ]. The revealed lipid-binding site in Vav2-SH2 was found to be adjacent to its highly cationic phosphotyrosine binding pocket.…”

“…The revealed lipid-binding site in Vav2-SH2 was found to be adjacent to its highly cationic phosphotyrosine binding pocket. However, NMR analysis suggested that the phosphotyrosine binding pocket might not be involved in lipid binding [ 59 ]. The lipid-binding site in the SH2 domain of Vav2 is quite similar to that of the Lck SH2 domain.…”

“…The binding of Vav2 to EphA2 is crucial for EphA2-mediated tumor angiogenesis [ 63 ]. Experiments employing tyrosine-phosphorylated peptides derived from the juxtamembrane region of EphA2 showed that Vav-SH2-EphA2 recognition is independent of phosphatidylinositol-4,5-bisphosphate or phosphatidylinositol-3,4,5-trisphosphate binding [ 59 ]. The weak phosphatidylinositol-4,5-bisphosphate or phosphatidylinositol-3,4,5-trisphosphate binding ability of Vav2 thus may contribute to its membrane recruitment [ 59 ].…”

“…Experiments employing tyrosine-phosphorylated peptides derived from the juxtamembrane region of EphA2 showed that Vav-SH2-EphA2 recognition is independent of phosphatidylinositol-4,5-bisphosphate or phosphatidylinositol-3,4,5-trisphosphate binding [ 59 ]. The weak phosphatidylinositol-4,5-bisphosphate or phosphatidylinositol-3,4,5-trisphosphate binding ability of Vav2 thus may contribute to its membrane recruitment [ 59 ]. Recent molecular dynamic simulations revealed that the proximal juxtamembrane domain along with the EphA2 kinase domain interacts with phosphatidylinositol-4,5-bisphosphate and that both domains induce the formation of phosphatidylinositol-4,5-bisphosphate nanoclusters in the membrane [ 64 , 65 ].…”