The Interaction Of Cytokeratin 18 And LRP16 Regulates Subcellular Localization Of LRP16

LRP16,a human gene cloned from the PBL(peripheral blood lymphocyte) of the healthy adult,is one of Macro Domain family member,which encoding protein sequence conservative and structure simple.Previous vitro study showed that LRP16 has interactions with transcription factors of nuclear receptor family memmbers including NF-κB,AR,ERβ,PPARα,PPARγ.Investigation certified LRP16 is a coen-reactivator of estrogen receptorα(ERα) and androgen receptor(AR).Function study verified LRP16 promoted oestrogenic hormonal dependent MCF-7 breast cancer cell generation and endometrial cancer Ishikawa cancer cell invasion.The role of LRP16 in estrogen hormone-dependent neoplasm genesis and development is significant.We screened LRP16 interaction proteins from cDNA library of MCF-7 human breast cancer cell line by yeast two-hybrid technology,the interaction between LRP16 and KRT18 was confirmed in yeast.KRT18 is one of cytoskeleton intermediate filament keratin family memmbers.Therefore,the interaction of LRP16 with KRT18 demand further confirment.Study certificated that differentiation related skeleton protein have interaction with many kinds of transcription factor or transcriptional coactivator in cytoplasm to detain neucleoprotein through interaction. Therefore,we suppose KTR18 could detain LRP16 in cytoplasm through interaction between them.The study can be divided into following two parts:一.The interaction between LRP16 and KRT18Objective:To further confirm the interaction of LRP16 with KRT18 and to identify interaction function epi-position of KRT18.Methods:1.Construct the recombinant expression vector of KRT18 full length and function structural domains.2.The interaction between LRP16 and KRT18 was confirmed by GST pull-down assay in vitro; In addition,we checked the interacting domains of both LRP16 and KRT18.3.The interaction between LRP16 and KRT18 was confirmed by co-immunoprecipitation analysis in vivo.Results:1.Successfully construct the recombinant expression vector of KRT18 and function construction domains.2.GST pull-down experiments confirmed interaction between LRP16 and C terminal region of KRT18 protein.3. Immuno-precipitation experiments verifield evidence of molecular interaction between KRT18 and endogenously expressed LRP16 proteins.Conclusions:1.These results propose reliable experimenta data for the molecular interaction of LRP16 with KRT18 protein in vitro and in vivo.2.GST pull-down experiments confirmed interaction between LRP16 and C terminal region of KRT18.3.C terminal region of LRP16 may interact with KRT 18.二.The influence on subcellular localization of LRP16 with KRT18Objective:To study the influence of Cytokeratin 18(KRT18) on subcellular distribution of LRP16 fusion protein from cell morphology,Western blot is initially used to further accredit the influence.Methods:1.The primarily structural sequence of LRP16 was recombined in pEGFP-N₁ vector,inserting in the N-termini of GFP(green fluorescent protein).2.LRP16-pEGFP was transfected,fluorescent signal was detected using fluorescentmicroscope.3.LRP 16-pEGFP was transiently cotransfected with Flag-pcDNA3-KRT18,subcellular distribution of LRP 16 was detected using fluorescent microscope.4.MCF-7 cell and Hela cell lines overexpressing and downregulating endogenous KRT18 expression were obtained,the expression in subcellular distribution of LRP16 was determined by Western-blot applying Nuclear/Cytosol extraction Kit. Results:1.The recombinant expression vector of LRP16-pEGFP was successfully constructed.2.The green fluorescene was inspired from cellular nucleolus in MCF-7 cells.LRP 16-pEGFP was located mainly in the cytoplasm of NIH3T3 cells.3.Contrasting the subcellular distribution of cotransfected cells treated with normal culture,KRT18 protein could mediate obviously LRP16 to distribute from cellular nucleus to cytoplasm.4. Expression of LRP16 of cytoplasm protein in MCF-7 and Hela cell lines overexpressing KRT18 was higher than that in cells negatively expressing KRT18.5.Conversely, downregulation of endogenous KRT18 expression by RNA interference decreased LRP16 protein level in cytoplasm.Conclusions:1.LRP 16 protein is principally distributed in the nuclei of epithelial tumour cells.2.KRT 18 could make LRP16 detain in cytoplasm through interacting with LRP16 in living cells.3.KRT18 may be very important regulating factor interacting with LRP 16 and affecting subcellular distribution of LRP 16 protein.