Study Of The Role And Mechanism Of RSU1 In Regulation Of The MEK-ERK Signaling

Cell-extracellular matrix(Cell-ECM)adhesion is crucial to tissue organization and cellular communication.Cells can perceive the environmental changes through cellextracellular matrix interaction,which leads to changes of cell behavior such as cell morphology,cell migration,cell proliferation,cell division,cell differentiation,cell apoptosis and so on.It is known that Cell-ECM detachment induces alterations of many intracellular signaling pathways,which inhibit cell proliferation and activate apoptosis,resulting in cell death known as “anoikis”.Anoikis is critical to tissue homeostasis,and cancer cells can become more aggressive by gaining resistance to anoikis.Integrin family proteins are critical ECM receptors on the cell membrane,and play key roles in mediating Cell-ECM interaction as well as sensing and transducing signals derived from ECM.Upon activation by binding to the ECM ligands,integrin recruits more than 180 intracellular proteins to the cell membrane through its intracellular domain.ILK,as a key signaling protein in integrin signaling,can form a ternary complex with parvin and PINCH known as IPP complex.Ras suppressor(RSU1)is another evolutionarily conserved focal adhesion.It has been demonstrated that PINCH interacts with RSU1,which recruits RSU1 to focal adhesion.How Cell-ECM detachment is linked to down-regulation of MEK-ERK signaling is however,incompletely understood.In this thesis,I found that RSU1-deficiency promotes MEK-ERK activation cell proliferation.Moreover,RSU1-deficiency inhibits cell spreading,cell migration and invasion.It was revealed that RSU1 is a negative regulatory factor of MEK-ERK signaling upon cell-ECM detachment.RSU1-deficiency inhibits cell-ECM detachment-induced decreases of MEK-ERK activation.Mechanistically,we have identified PHB2 as a new binding partner of RSU1,which regulates MEK-ERK activation positively on lipid rafts.Using co-immunoprecipitation,pull-down,and FRET assays,I have proved the interaction between RSU1 and PHB2.Interestingly,the RSU1-PHB2 interaction is enhanced upon cell-ECM detachment.It was also revealed that RSU1 is distributed on lipid rafts and the association of RSU1 with lipid rafts is increased upon cell-ECM detachment.In addition,I have mapped a major RSU1-binding site to PHB2 amino acids 150-206 in the C-terminal region of the PHB domain.This PHB2(aa 150-206)fragment,which shows higher affinity to RSU1 than full-length PHB2 to RSU1,plays a dominant negative role by disrupting the interaction between RSU1 and PHB2.Overexpression of the PHB2(aa 150-206)fragment,like RSU1-deficiency,inhibits the decreases of MEK-ERK activation induced by cell-ECM detachment,while overexpression of RSU1-C-term,which is unable to interact with PHB2,could not mediate MEK-ERK activation.Together,these results strengthen the role of RSU1-PHB2 interaction in regulating MEK-ERK activation.It is known that RSU1 is recruited to focal adhesion via interaction with PINCH1.PINCH1 is implicated in regulation of the MEK-ERK signaling.Therefore,the role of RSU1 in PINCH1-mediated regulation of the MEK-ERK signaling pathway was also investigated.It was revealed that RSU1 is indeed involved in the suppression of PINCH1-mediated MEK-ERK activation.Moreover,using the RSU1-Y140 K mutant,which is unable to interact with PINCH1,I have demonstrated that the MEK-ERK signaling mediated by RSU1-PHB2 axis is independent of RSU1-PINCH1 interaction.In conclusion,this thesis shows a mechanism of RSU1-PHB2 axis by which MEK-ERK activation is regulated,which is also supported by cells grown in 3D culture.This thesis shows explicitly how RSU1-PHB2 axis regulated MEK-ERK activation,shedding light on our understanding of integrin signaling and cancer development and metastasis.This may also provide potential new therapeutic targets for cancer treatment.