PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodelling

Abstract

Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST is a central regulator of actin cytoskeletal dynamics. Here we demonstrate that an N‑terminal fragment of PTP-PEST, generated through ephrinA5-triggered, spatially confined cleavage by caspase‑3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation similar to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and via its direct action as PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling.