Abstract
Eph receptor tyrosine kinases play a key role in cell-cell communication. Lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered understanding of their signaling mechanisms. Here, we use integrative structural biology to investigate the structure and dynamics of the EphA2 intracellular region. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling involving serine/threonine phosphorylation of the linker connecting its kinase and SAM domains. We show that accumulation of multiple linker negative charges, mimicking phosphorylation, induces cooperative changes in the EphA2 intracellular region from more closed to more extended conformations and perturbs the EphA2 juxtamembrane segment and kinase domain. In cells, linker negative charges promote EphA2 oligomerization. We also identify multiple kinases catalyzing linker phosphorylation. Our findings suggest multiple effects of linker phosphorylation on EphA2 signaling and imply that coordination of different kinases is necessary to promote EphA2 non-canonical signaling.
Highlights
Eph receptor tyrosine kinases play a key role in cell-cell communication
How the different parts of the Eph receptor intracellular region— including the juxtamembrane segment, kinase domain, linker and sterile alpha motif (SAM) domain—are arranged and function together has remained an open question, in part due to lack of structural information that extends beyond single domains and adjacent regions[14,37,53]
Our data, based on introduction of glutamic acid as a proxy for serine/threonine phosphorylation, suggest a model for the conformational dynamics that underlie EphA2 non-canonical signaling induced by cumulative phosphorylation of the kinase-SAM linker
Summary
Eph receptor tyrosine kinases play a key role in cell-cell communication. Lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered understanding of their signaling mechanisms. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling involving serine/threonine phosphorylation of the linker connecting its kinase and SAM domains. The intracellular portion of the Eph receptors includes a juxtamembrane segment, the tyrosine kinase domain, a linker, a sterile alpha motif (SAM) domain, and a short C-terminal tail containing a PDZ domain-binding motif (Fig. 1a). Despite its profound significance in cancer, the mechanism of non-canonical signaling is not well understood[22,26] It is unknown how phosphorylation of S897 in the linker connecting the EphA2 kinase and SAM domains regulates EphA2 signaling. Our findings support a model in which multiple negative charges introduced by linker phosphorylation cooperate to promote an ensemble of open conformations of the EphA2 intracellular region that mediate non-canonical signaling. We find that multiple EphA2 kinase-SAM linker residues can be simultaneously phosphorylated by different kinases, consistent with the notion that cumulative linker phosphorylation is important for EphA2 non-canonical signaling
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