Minglyanna Surudoi

EphB6 is an understudied ephrin receptor tyrosine pseudokinase that is downregulated in multiple types of metastatic cancers. Unlike its kinase-active counterparts which autophosphorylate and transmit signals upon intercellular interaction, little is known about how EphB6 functions in the absence of intrinsic kinase activity. Here, we unveil a molecular mechanism of cell-cell interaction driven by EphB6. We identify ephrinB1 as a cognate ligand of EphB6 and show that in trans interaction of EphB6 with ephrinB1 on neighboring cells leads to the formation of large co-clusters at the plasma membrane. These co-clusters exhibit a decreased propensity towards endocytosis, suggesting a unique characteristic for this type of cell-cell interaction. Using lattice light-sheet microscopy, 3D structured illumination microscopy and cryo-electron tomography techniques, we show that co-clustering of EphB6 and ephrinB1 promotes the formation of double-membrane tubular structures between cells. Importantly, we also demonstrate that these intercellular structures stabilize cell–cell adhesion, leading to a reduction in the invasive behavior of cancer cells. Our findings rationalize a role for EphB6 pseudokinase as a tumor suppressor when interacting with its ligands in trans.

PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate the molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding and signaling via Grb2. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

EphB6 is an understudied ephrin receptor tyrosine pseudokinase, the expression of which is downregulated in multiple types of metastatic cancers. Compared to its kinase-active counterparts, how EphB6 regulates signal transduction in the absence of intrinsic kinase activity remains unknown. Here, we unveil the molecular details of key signal transduction mechanisms driven by EphB6. We identify ephrinB1 as a cognate ligand of EphB6 and demonstrate EphB6’s ability to form extensive co-clusters at the plasma membrane upon binding ephrinB1 in trans across cell-cell contacts, which are resistant to endocytosis. Using super resolution microscopy, correlative light and electron microscopy and cryo-electron tomography, we reveal that co clustering of EphB6 and ephrinB1 promotes the formation of unprecedented double membrane tubular structure interconnecting cells. Importantly, phenotypically, co-clustering of EphB6 and ephrinB1 and the resulting tubular structures suppress cancer cell invasion by prolonging cell-cell contacts, rationalising a role for EphB6 pseudokinase as a tumor suppressor when interacting with its ligands in trans.

PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate new molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

The pseudokinase scaffolds PEAK1 and PEAK2 are implicated in cancer cell migration and metastasis. We characterized the regulation and role of the third family member PEAK3 in cell signaling. Similar to PEAK1 and PEAK2, PEAK3 formed both homotypic and heterotypic complexes. In addition, like PEAK1, it bound to the adaptors Grb2 and CrkII. However, unlike PEAK1 and PEAK2, homodimerized PEAK3 also interacted with the ARF GTPase-activating protein ASAP1, the E3 ubiquitin ligase Cbl, and the kinase PYK2. Dimerization and subsequent phosphorylation on Tyr ²⁴ , likely by a Src family kinase, were required for the binding of PEAK3 to Grb2 and ASAP1. Interactions with Grb2, CrkII, ASAP1, Cbl, and PYK2 exhibited contrasting dynamics upon cell stimulation with epidermal growth factor (EGF), in part due to PEAK3 dephosphorylation mediated by the phosphatase PTPN12. Overexpressing PEAK3 in mesenchymal-like MDA-MB-231 breast cancer cells enhanced cell elongation in a manner dependent on PEAK3 dimerization, and manipulation of PEAK3 expression demonstrated a positive role for this scaffold in regulating cell migration. Overexpressing PEAK3 in PEAK1/2 double-knockout MCF-10A breast epithelial cells enhanced acinar growth, impaired basement membrane integrity, and promoted invasion in three-dimensional cultures, with the latter two effects dependent on the binding of PEAK3 to Grb2 and ASAP1. PEAK1 and PEAK2 quantitatively and temporally influenced PEAK3 function. These findings characterize PEAK3 as an integral, signal-diversifying member of the PEAK family with scaffolding roles that promote cell proliferation, migration, and invasion.