PTP1B regulates Eph receptor function and trafficking

Changes in protein tyrosine phosphatase 1B expression affect duration and amplitude of EphA3 phosphorylation and cell surface concentration

Regulation of EphA3 receptor tyrosine kinase signalling by protein tyrosine phosphates

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

Regulation of EphA3 receptor tyrosine kinase signalling by protein tyrosine phosphates

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

Elevated protein tyrosine phosphatase activity provokes Eph/ephrin-facilitated adhesion of pre-B leukemia cells

Signaling by Eph receptors and cell-surface ephrin ligands modulates adhesive cell properties and thereby coordinates cell movement and positioning in normal and oncogenic development. While cell contact–dependent Eph activation frequently leads to cell-cell repulsion, also the diametrically opposite response, cell-cell adhesion, is a probable outcome. However, the molecular principles regulating such disparate functions have remained controversial. We have examined cell-biologic mechanisms underlying this switch by analyzing ephrin-A5–induced cell-morphologic changes of EphA3-positive LK63 pre-B acute lymphoblastic leukemia cells. Their exposure to ephrin-A5 surfaces leads to a rapid conversion from a suspended/nonpolarized to an adherent/polarized cell type, a transition that relies on EphA3 functions operating in the absence of Eph-kinase signaling. Cell morphology change and adhesion of LK63 cells are effectively attenuated by endogenous protein tyrosine phosphatase (PTP) activity, whereby PTP inhibition and productive EphA3-phosphotyrosine signaling reverse the phenotype to nonadherent cells with a condensed cytoskeleton. Our findings suggest that Eph-associated PTP activities not only control receptor phosphorylation levels, but as a result switch the response to ephrin contact from repulsion to adhesion, which may play a role in the pathology of hematopoietic tumors. Copyright © 2008 by American Society of Hematolog

Eph receptor function is modulated by heterooligomerization of A and B type Eph receptors.

Eph receptors interact with ephrin ligands on adjacent cells to facilitate tissue patterning during normal and oncogenic development, in which unscheduled expression and somatic mutations contribute to tumor progression. EphA and B subtypes preferentially bind A- and B-type ephrins, respectively, resulting in receptor complexes that propagate via homotypic Eph–Eph interactions. We now show that EphA and B receptors cocluster, such that specific ligation of one receptor promotes recruitment and cross-activation of the other. Remarkably, coexpression of a kinase-inactive mutant EphA3 with wild-type EphB2 can cause either cross-activation or cross-inhibition, depending on relative expression. Our findings indicate that cellular responses to ephrin contact are determined by the EphA/EphB receptor profile on a given cell rather than the individual Eph subclass. Importantly, they imply that in tumor cells coexpressing different Ephs, functional mutations in one subtype may cause phenotypes that are a result of altered signaling from heterotypic rather from homotypic Eph clusters

Publisher Correction: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion

An Amendment to this paper has been published and can be accessed via a link at the top of the paper

Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse; 1; that is driven by chemotherapy-resistant leukaemic stem cells (LSCs); 2,3; . LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice; 4; . However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity; 5; , which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D-a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells; 6-9; -are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo