STYK1 Regulates The Formation Of Autophagy Complex And Promotes Autophagy

Autophagy is a regulated self-eating process that eliminates the damaged or inactivated proteins and organelles and other substrates in the cell through the autophagosom-lysosomal degradation pathway,it plays an important role in maintaining homeostasis in the intracellular environment.The regulation process of autophagy is highly complex and elaborate.So far,41 autophagy-related genes(ATG)have been identified to participate in the process.Among them,more than 30 autophagy-related genes are crucial for the formation of autophagosomes(the core initiation process of autophagy).However,the regulatory mechanism of autophagy initiation process is still un Clear,especially the formation of autophagosomes mediated by the autophagy-specific Class III phosphatidylinositol 3-kinase complex I(PtdIns3K-C1).In this study,western blotting was used to detect the expression levels of autophagy marker LC3 protein and autophagy substrate p62 protein.It was found that in Hep G2 lung cancer cell lines,specific knockdown or exogenous overexpression of tyrosine kinase STYK1 inhibited or promoted the level of autophagy.Immunofluorescence experiments showed that in Hep G2 cell lines,knocked down STYK1 gene,the point aggregates of LC3 protein were reduced and the formation of autophagosomes and autophagosomes was inhibited,in addition,knocked down STYK1 gene in Hela cell lines,and the formation of autophagy vesi Cles was significantly inhibited.These phenomena suggest that STYK1 promotes the process of autophagy.These phenomena indicate that STYK1 promotes the process of autophagy.The Co-immunoprecipitation and GST-pull down results showed that STYK1 protein binds to PtdIns3K-C1 complex-related protein(ATG14-BECN1-PI3KC3-PIK3R4)in Hela cell line,Immunofluorescence also showed that STYK1 protein colocated with ATG14-BECN1-PI3KC3-PIK3R4 and PtdIns3K-C1 complex downstream key effectors ZFYVE1 and WIPI1 in Hela cells.Immunofluorescence also showed that STYK1 protein in Hela cells co-localized with ATG14-BECN1-PI3KC3-PIK3R4 and the key downstream effectors ZFYVE1 and WIPI1 of the PtdIns3K-C1 complexes,in addition,the overexpression of STYK1 can promote the puncta number of ATG14,ZFYVE1 and WIPI1 proteins.These results confirmed that STYK1 protein localized on the autophagosome and promoted autophagy by stimulating the formation of autophagosomes.Finally,we also found that STYK1 protein was overexpressed in Hela cell line,which enhanced the interaction between PI3KC3,ATG14 and BECN1,and inhibited the interaction between BECN1 and the negative autophagy regulators RUBCN and BCl2.And knocking down STYK1 gene inhibited the activity of PtdIns3P in Hela cell line,which further confirmed that STYK1 protein promotes autophagy by promoting the assembly of ATG14-BECN1-PI3KC3 protein complex and PtdIns3P kinase activity specifically.In summary,this study revealed that the STYK1 protein(serine/threonine/tyrosine kinase 1),a member of the receptor tyrosine kinase(RTKs)family,serves as a positive regulator of autophagy.It promotes autophagy by regulating the assembly and function of PtdIns3K-C1 complex.Which deepens the understanding of the regulation of PI3 K signaling pathway function and provides new insights into the induction of autophagy from the perspective of RTK signaling regulation.