Studies Of Relationship Between PYK2and Adaptor Leupaxin And Their Roles In T Cell Activation And Motility

ObjectiveTo prove the relationship between PYK2and adaptor Leupaxin and the roles of thiscomplex in T cell activation and motilityMethodsTo illustrate the probability of the existence of the complex of PYK2andLeupaxin(LPXN), Fibronectin (FN) stimulated JurkatE6cell, which is an acute T leukemiacell line, was chosen to be the model. Fluorescent tagged proteins were overexpressed inJurkat E6and their locations were observed via fluorescent microscope and confocalimmunofluorescent microscope, also immunoprecipitation assay was applied to find thecombination of these proteins.In order to know the mechanism of the effect between PYK2and LPXN, JurkatE6was stimulated by Fibronectin(FN) and Human SDF-1(Hu-SDF-1) and then therelationship between proteins and the variance of tyrosine phosphorylation in PYK2(endogenesis and ectogenesis) were proved by using immunoprecipitation assay. Toobserve the activation, adhesion and migration after knockdown of LPXN with RNAinterference and overexpress PYK2and LPXN with transient transfection. At the same time,changes in different tramsfected cells could be observed from Transwell and adhesion assayand the variances of motility and polarization in these cells could be seen in Time LapseCell Video. More importantly, we also demonstrate the diversity of the ability of cell movement in Peripheral Blood Lymphocyto(PBL) when LPXN silenced.ResultsPossibility of existence of PYK2/LPXN complex and their combination:(1) Thelocalization of LPXN was shifted from nucleus to cytoplasm after stimulated by FN,usually LPXN was localized in nuclear but it can switch to cytoplasm membrane andcolocalized with PYK2in focal adhesion site(s), this could be directly found under thefluorescent microscope and immunofluorescent confocal microscope when overexpressPYK2and LPXN with fluorescent tagged plasmids.(2) Furthermore, immunoprecipitationassay showed that PYK2and LPXN might bind to each other and facilitate their interaction,enhance the possibility of the existence of this complex.The relationship between proteins:(1) Phosphorylation of LPXN was reinforcedunder the stimulation of FN. And the tyrosine phosphorylation of PYK2in JurkatE6wasenhanced by the time of FN and/or Hu-SDF-1stimulation and the phosphorylation peakwas observed during2min to5min when stimulating then decrease gradually after10min.(2) Meanwhile, immunoprecipitaion assay also illustrate that beside get phosphorylatedautomatically, the tyrosine phosphorylation of PYK2could be reinforced by LPXN.Augmentation of this phosphorylation of PYK2could be seen when LPXN wasoverexpress in Jurkat E6. In the contrast, PYK2’s tyrosine phosphorylation was decreasedwhen LPXN get silenced and the phosphorylation was also not able to apparently detectneither in ectogenesis PYK2in JurkatE6. These phenomenons demonstrate that LPXN isessential to enhance PYK2’s tyrosine phosphorylation.(3) LPXN can augment adhesionand to weaken migration of JurkatE6cells under stimulation of FN and Hu-SDF-1inTranswell and Adhesion assay. But when LPXN get silenced the adherent positive cells andtheir adhesion ability was decreased whilst the migrated positive cells and their migrationcapability was raise up. However, cell adhesion was enhanced and migration was decreasedwhen LPXN get overexpressed. The cell movement can be easily tracked in LPXN silenced cells while nearly no cell trajectory was observed when LPXN get overexpressed.(4) Theweaker adhesion and stronger migration in LPXN knockdown cells were proved in theTime Lapse Cell Video. The movement of LPXN silenced cells was much faster whenstimulated with FN and Hu-SDF-1while the phenomenon is opposite when LPXN getoverexpressed. Same conclusions were observed in PBL (Pheripheral Blood Lymphocyte).ConclusionPYK2and Leupaxin might combine and interact with each other. Besides getphosphorylated automatically, PYK2’s tyrosine phosphorylation could be mediated andaugmented by LPXN, no matter endogenesis or ectogenesis cen enhance cell adhesion andattenuate its movement.