Regulation of EphA3 receptor tyrosine kinase signalling by protein tyrosine phosphates
Creators: name: Nievergall, Eva
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Eph receptor tyrosine kinases (RTKs) and their membrane-bound ephrin ligands control a multitude of processes during embryonic development, such as cell positioning, tissue patterning and organogenesis. While showing limited expression and activity in normal adult tissues Ephs and ephrins re-emerge during tumour progression and metastasis. Eph/ephrin interactions typically affect cell adhesion and migration, where diametrically¬opposing cellular responses, ranging from cell-cell adhesion and spreading to cytoskeletal collapse and cell-cell segregation, can be promoted. The ultimate outcome is strictly guarded by the composition of Eph/ephrin signalling clusters and cytoplasmic control of Eph kinase activity. While structural aspects and mechanisms of Eph activation and clustering have been extensively studied, the negative regulation of Eph R TK. activity by counter-acting protein tyrosine phosphatases (PTPs), well-established major modulators of RTK. signal transduction, remained largely unexplored. Here, we used the EphA3 receptor, identified by cancer genome-profiling of somatic mutations as a candidate cancer gene and currently undergoing clinical trials as an anti¬cancer target, as a paradigm to conclusively study the role of PTPs in controlling Eph activity and function in normal and tumour cells. Initially, we ascertained the essential role of PTPs during EphA3 signalling by establishing that high endogenous PTP activity in LK63 pre-B leukaemia cells, keeping EphA3 phosphorylation at a negligible level, switches the cellular response to epbrinAS contact from repulsion to adhesion. Further studies identified two cytosolic PTPs, previously not implicated in Eph signalling, as critical modulators of Eph kinase activity and cytoskeletal plasticity. PTPIB, a prototypic regulator of several growth factor receptors, directly controls EphA3 kinase activity and phosphorylation in normal and cancer cells thereby critically affecting the outcome of EphA3/epbrinAS-governed cytoskeletal contraction and cell-cell segregation. Interestingly, functional single-cell imaging analysis revealed that the ER-resident PTPIB targets the phosphorylated receptor at the cell surface -in particular at sites of cell-cell contacts, where it controls endocytosis of activated Ephs, as well as on endosomes. To our surprise, PTP-PEST, a known regulator of focal adhesion turnover and cell motility, also decelerates EphA3 phosphorylation, indicating a tight link between cytoskeletal plasticity and Eph receptor activation at the plasma membrane. By developing a unique detergent-free cell fractionation strategy that allows recovery of intact EphA3 plasma membrane signalling clusters, we show that spatially confined ephrinA5-induced caspase-3 activation yields an N-terminal fragment of PTP-PEST, which associates with activated EphA3 at the plasma membrane together with a range of actin cytoskeletal and focal adhesion proteins. As caspase inhibition rescues PTP-PEST -attenuated EphA3 phosphorylation, this cleavage is critical for PTP-PEST's negative regulatory function. In summary, we show here that for EphA3, as for other protein tyrosine kinases, a finely tuned balance between tyrosine phosphatase and kinase activities is essential for its function. Importantly, the control of EphA3 cell surface concentration by both PTPIB and PTP-PEST via distinct mechanisms provides novel insights in the regulation of Eph receptor clustering and signal activation. The established involvement of PTPIB and EphA3, and the emerging role of PTP-PEST, in tumour growth and progression imply a significant relevance of these regulatory circuits in oncogenesis and metastasis.