Study On The Mechanism Of I-BAR Family Proteins In Cancer Cell Signaling Pathway

The inverse BAR(I-BAR)domain family proteins were reported participating membrane deformation and associating with a range of physiological and pathological process.Missing-in-metastasis(MIM),is a putative tumor suppressor gene since it is oftenly dysregulated in various advanced cancers.The protein it encodes is MIM protein,which is a prototype of I-BAR family.In this study,MIM knockout mouse strain was established,based on which we found MIM-/-bone marrow(BM)cells exhibited an elevated chemotaxis towards SDF-1(chemokine stromal-derived factor 1)comparing to wild type cells.Besides,constitutive activation of p38,Rac,CDC42 and impairment in CXCR4 internalization were also detected.MIM-/-BM cells including HSPCs which were transplanted into lethally irradiated mice exhibited improved homing efficiency to bone marrow.What's more,this process was dependent on the phosphorylation of p38 MAP kinase.Therefore,MIM regulates the interaction of bone marrow cells with their microenvironment by modulating the SDF-1/CXCR4 axis and the downstream signaling pathway.HeLa cells which were stably expressing MIM-GFP(or GFP)were established to illustrate the mechanism for the phenomenon MIM modulating CXCR4.It was found that overexpression of MIM suppressed cellular response to SDF-1 and promoted CXCR4 internalization which was correlated with enhanced CXCR4 ubiquitination and faster degradation.Besides,it was detected that MIM could directly bind E3 ubiquitin ligase AIP4 which was well established being involved in CXCR4 Ubiquitination.Under this mechanism,MIM,AIP4 and CXCR4 formed a complex in response to SDF-1.Of note,we found that binding of AIP4 depends on the proline rich domain(PRD)especially the PPLP sequence located in MIM's C-terminal,PPLP within PRD is a theoretical target of AIP4's WW domain.Deletion of either PRD or PPLP sequence could impair the association of MIM and AIP4,and further abolish the interaction between MIM and CXCR4.In addition,it was demonstrated that MIM could promote multi-vesicular bodies(MVB)' bio-generation through interacting with Rab7 via its I-BAR domain.As a result,endosomal sorting of CXCR4 to late endosome from early endosome after internalized form cell membrane was facilitated,which lead to more CXCR4 was transported into lysosome for degradation.In parallel with binding to Rab7,MIM promoted MVBs formation through forming a complex with ESCRT-0 subunit upon SDF-1 stimulation.To compare the functins of I-BAR proteins in cancer cell,another I-BAR protein IRTKS was also investigated for its role on modulating cancer cell migration.Different with MIM,over-expression of IRTKS improved chemotaxis to serum along with upregulated activation of MAPK Erkl/2 and p38,as well as activation of small GTPases Racl and CDC42.Of interest,the SH3 domain within IRTKS's C-terminal distinguishes the function of IRTKS and MIM since deleting this domain leading to abolished activity of IRTKS.Finally,it was found that both IRTKS and MIM made great contribution to cell morphology.IRTKS overexpression results in cell polarization and formation of lamellipodia,which dependent on the co-operation of its N-and C-terminals;On the other hand,MIM protein facilitated the generation of lamellipodia.Since SDF-1/CXCR4 axis,IRTKS and MIM are dysregulated in cancerous cells,the data in this study evidenced a new mechanism which briges an oncogenic process to an endocytic pathway.