Cellular Function Study Of The Interaction Between Parkinson’s Disease-Associated Protein LRRK2 And PAKs Proteins

Objective:To study the function of the interaction between LRRK2 and type II PAKs in cells,and how LRRK2 mutation affected the interaction.To clarify the interaction mechanism and internal relationship between LRRK2 and PAKs from the perspective of cell biology,providing a new theoretical basis for understanding the pathogenic mechanism of LRRK2 in Parkinson’s disease.Methods:The recombinant protein expression plasmids were constructed by molecular cloning technology,including LRRK2 full-length protein with HA tag and GFP fluorescent tag,LRRK2_RCKW protein with Cy Pet fluorescent tag,PAK5 and PAK6proteins with Myc tag,GFP and YPet fluorescent tag.The plasmids were transfected into a mammalian cell line（HEK293T）to transiently express the proteins.The interaction between LRRK2 and PAKs proteins and the extent of their interaction were determined by FRET.The protein expression level and kinase activity were detected by Western blot.The localization of the two proteins in mitochondria was examined by Mito-Tracker Red CMXRos.Results:The FRET assay showed that LRRK2 interacted with PAK6 and PAK5in cells.Compared with the wild type,pathogenic and protective mutations in the Roc domain of LRRK2 enhanced its interaction with PAK5 and PAK6（P<0.05）.Co-expression of LRRK2 and PAKs in HEK293T cells did not significantly affect the phosphorylation level of PAKs.At the same time,there was no significant effect on the phosphorylation level of LRRK2 at Ser1292 in the endogenous LRRK2 and the system overexpressing wild-type LRRK2 and LRRK2 R1441C/H.However,the phosphorylation of LRRK2 at Ser1292 was decreased in the LRRK2 R1398H overexpressed system.In contrast,the phosphorylation of LRRK2 at Ser1292 was increased in the LRRK2 N1437H overexpressed system.Pearson correlation coefficient analysis after fluorescent probe staining showed that PAK6 and LRRK2 co-localized in mitochondria with stronger signals than PAK5.After the co-expression of PAKs and LRRK2 in cells,the distribution pattern of LRRK2 in cells was changed from diffusion to scattered aggregation distribution.Overexpression of wild-type LRRK2 and R1398H and R1441H mutants enhanced the co-localization fluorescence signal of PAK5 and mitochondria,while N1437H and R1441C mutants did not change the localization of PAK5 in mitochondria.Overexpression of LRRK2（wild type and mutant）did not significantly change the co-localization fluorescence signal between PAK6 and mitochondria.Conclusion:LRRK2 could interact with PAK6 and PAK5 in cells.The co-expression of LRRK2 and PAKs in cells can change the distribution pattern of LRRK2in cells.PAK6 and LRRK2 were localized in the mitochondria,but PAK5 was not.Pathogenic and protective mutations in the Roc domain of LRRK2 could enhance its interaction with PAKs and change the localization of PAK5 in the mitochondria,but not PAK6.