The Regulation Of E-Cadherin-Mediated Cell Adherent Junctions By The Activation Of Rap1and H-Ras

Adherent junction is the epithelial adhesion structure mediated by E-cadherins,of which the extracellular domain could trans-interact with the same that on theoppsite cells, while the intracellular domain is linked to the actin cytoskeletonthrough series of catenins including p120-catenin, β-catenin and so on. Adherentjunctions connect up adjacent cells in turn, maintaining the normal morphology andpolarity of epithelial cells, and participate in the intercellular and intracellular signaltransduction, inhibiting tumorigenesis and metastasis. Ras family small G proteinsact as the important regulators of E-cadherin. Rap1can promote the formation andlocalization of adherent junctions mediated by E-cadherins, but in these processesthe downstream signaling pathways of Rap1are still unknown. It has been reportedthat depressed activity of Ras could enhance the expression of E-cadherin, andmouse embryonic fibroblasts lack of H-Ras shows a lower proliferation andmigration capacity, but whether H-Ras could regulate E-cadherin has not beenconfirmed yet. To address whether Rap1and H-Ras could collaborately orantagonisticly regulate the adherent junction formation mediated by E-cadherin, weapplied calcium switch to induce adherent junction reformation. The activation stateof Rap1and H-Ras during adherent junctions disruption and reformation wasdetected through the GST-Pull down technique. Our results showed that both Rap1and H-Ras in MCF-7cells were activated during adherent junctions disruption byCa²⁺depletion. Furthermore, Rap1and H-Ras interacting proteins during adherentjunctions disruption and reformation were identified through the Co-IP technique.By using FRET techniques the positions where Rap1and H-Ras are activated in thecell were detected. Totally, our results revealed that the activation of both Rap1andH-Ras was related to adherent junction reformation during Ca²⁺switch assay. Rap1downstream effector candidates mainly consist of cytoskeletal proteins, cytoskeletalregulatory proteins and small G protein GAPs and H-Ras downstream effectors mayalso include some cytoskeletal proteins. Rap1initial activation level around thenuclear is above that near the membrane, H-Ras activation level either on themenbrane or in the cytoplasm increases during adherent junctions disruption anddecreases during adherent junction reformation.Our results provided cues to understand the functional relations between Rap1and H-Ras, especially in the molecular regulation mechanism of cell adherentjunctions mediated by E-cadherins.