Biology subjects

Banerjee, S. L.

Publications and source records attributed to Banerjee, S. L..

3 recordsLinked to original sources

bioRxiv2024

EPHA1 and EPHB4 tyrosine kinase receptors regulate epithelial morphogenesis

Organ formation and homeostasis require the coordination of cell-cell adhesion, epithelial cell polarity and orientation of cell division to organize epithelial tissue architecture. We have previously identified proximity protein networks acting downstream of members of the EPH family of tyrosine kinase receptors and found within these networks an enrichment of components associated with cell morphogenesis and cell-cell junctions. Here, we show that two EPH receptors, EPHA1 and EPHB4, are localized to the basolateral domain of Caco-2 cells in spheroidal cultures. Depletion of either EPHA1 or EPHB4 disrupts spheroid morphogenesis, without affecting cell polarity, but via randomizing mitotic spindle orientation during cell division. Strikingly, EPHA1 and EPHB4 exert this function independently of their catalytic activity but still requiring EFN ligand binding. Consistent with this, the most abundantly expressed EPHB4 ligand in Caco-2 cells, EFNB2, is also compartmentalized at the basolateral domain in spheroids, and is required for epithelial morphogenesis. Taken together, our data reveal a new role for EPHRs in epithelial morphogenesis.

cell biology↗

bioRxiv2023

Ubiquitin ligase and signalling hub MYCBP2 is required for efficient EPHB2 tyrosine kinase receptor function

Eph receptor tyrosine kinases participate in a variety of normal and pathogenic processes during development and throughout adulthood. This versatility is likely facilitated by the ability of Eph receptors to signal through diverse cellular signalling pathways: primarily by controlling cytoskeletal dynamics, but also by regulating cellular growth, proliferation, and survival. Despite many proteins linked to these signalling pathways interacting with Eph receptors, the specific mechanisms behind such links and their coordination remain to be elucidated. In a proteomics screen for novel EPHB2 multi-effector proteins, we identified human MYC binding protein 2 (MYCBP2 or PAM or Phr1). MYCBP2 is a large signalling hub involved in diverse processes such as neuronal connectivity, synaptic growth, cell division, neuronal survival, and protein ubiquitination. Our biochemical experiments demonstrate that the formation of a complex containing EPHB2 and MYCBP2 is facilitated by FBXO45, a protein known to select substrates for MYCBP2 ubiquitin ligase activity. Formation of the MYCBP2-EPHB2 complex does not require EPHB2 tyrosine kinase activity and is destabilised by binding of ephrin-B ligands, suggesting that the MYCBP2-EPHB2 association is a prelude to EPHB2 signalling. Paradoxically, the loss of MYCBP2 results in increased ubiquitination of EPHB2 and a decrease of its protein levels suggesting that MYCBP2 stabilises EPHB2. Commensurate with this effect, our cellular experiments reveal that MYCBP2 is essential for efficient EPHB2 signalling responses in cell lines and primary neurons. Finally, our genetic studies in C. elegans provide in vivo evidence that the ephrin receptor VAB-1 displays genetic interactions with known MYCBP2 binding proteins. Together, our results align with the similarity of neurodevelopmental phenotypes caused by MYCBP2 and EPHB2 loss of function, and couple EPHB2 to a signaling effector that controls diverse cellular functions.

cell biology↗

bioRxiv2020

Proximity proteomics reveal EPH tyrosine kinase receptors' regulation of cell polarity and identify Par3 scaffold protein as a novel effector of EPH signaling

The EPH family is the largest among receptor tyrosine kinases (RTKs) in humans. In contrast to other RTKs, EPH receptors (EPHRs) cognate ligands, ephrins, are tethered to the cell surface. This results in EPHR-ephrin signaling being mainly involved in short-range cell-cell communication events that regulate cell adhesion, migration and tissue boundary formation. Although EPHRs functions have been broadly studied, the molecular mechanisms by which they control these processes are far from being understood. To address this, we sought to identify new effector proteins acting downstream of EPHRs and determine their role in EPHR-regulated functions. To unravel EPHR-associated signaling complexes under native conditions, we applied a mass spectrometry-based approach, namely BioID proximity labeling. We obtained a composite proximity network from EPHA4, -B2, -B3 and -B4 receptors that comprises 395 proteins, most of which were not previously linked to EPH signaling. A gene ontology and pathway term analysis of the most common candidates highlighted cell polarity as a novel function associated with EPHR activity. We found that EPHA1 and EPHB4 expression is restricted to the basal and lateral membrane domains in polarized Caco-2 3D spheroidal cell cultures. We further discovered that their depletion impairs the compartmentalized distribution of polarity proteins as well as overall spheroid morphogenesis. Moreover, we examined the contribution of a number of candidates, selected from EPHR proximity networks, via loss-of-function in an EPHR-dependent cell segregation assay. We found that depletion of the signaling scaffold PAR-3 blocks cell sorting. We also delineated a signalling complex involving the C-terminal SRC kinase (CSK), whose recruitment to PAR-3 complexes is dependent on EPHR signals. Our work sheds a new light on EPHR signaling networks and describes conceptually novel the mechanisms by which EPHRs signal at the membrane to contribute to the regulation of cellular phenotypes.

molecular biology↗