The Role Of Casein Kinase 1 Family Member CK1? In Macroautophagy Pathway

Autophagy refers to the process in which the damaged proteins or organelle are engulfed by the double-membranous autophagosomes and transported to lysosomes(animals)or vacuoles(yeasts and plants)for degradation and recycling.The CK1 family of serine/threonine kinases regulates diverse cellular processes,including membrane transport,DNA repair,apoptosis and autophagy,through binding to and phosphorylation different protein substrates.It has been reported that casein kinase?1 ?(CK1?)suppresses tumour growth by activating an autophagy-regulating,tumour-suppressive PTEN/AKT/FOXO3a/Atg7 axis.And in Saccharomyces cerevisiae,Hrr25,a casein kinase 1 family member,regulates macroautophagy by phosphorylating Sec24,and phosphorylates autophagy cargo receptor proteins during selective autophagy.However,the role of CK1? isoform in macroautophagy remains unclear.To explore the role of CK1? in macroautophagy,CK1? was first knocked down with si RNA interference technology,and the levels of autophagy substrate p62 and autophagy marker LC3-II in CK1?-depleted He La cells were detected by Western blot and immunofluorescence.The results showed that autophagy substrate p62 was accumulated,with increased autophagy-related protein LC3-II level in CK1?-depleted cells compared to control cells under starvation conditions,indicating that macroautophagy pathway was blocked after LC3-II lipidation.p62 and LC3-II were accumulated,with bafilomycin A1 treatment(inhibiting the fusion of autophagosome and lysosome)under starvation conditions,suggesting that autophagy pathway was blocked prior to the fusion of autophagosome and lysosome.Collectively,these findings indicate that autophagy pathway was blocked after LC3 lipidation and before the fusion of autophagosome and lysosomes in CK1?-depleted cells.Subsequently,the specific action site of CK1? in the starvation-induced macroautophagy pathway was further investigated.Numbers of autophagosomes and autophagolysosomes were detected with RFP-GFP-LC3 tandem fluorescence reporter plasmid.The findings showed the increase of autophagosomes and the decrease of autophagolysosomes in CK1?-depleted cells,indicating that autophagy defects occur before the fusion of autophagosome and lysosome,resulting in a large accumulation of autophagosomes.A protease K protection assay was then conducted to determine whether autophagy defects occurred before or after autophagosome closure.And the results show that neither p62 nor GFP-LC3 were protected by autophagosomesin CK1?-depleted cells,exhibiting the increased sensitivity of p62 and GFP-LC3 to proteinase K.Defect in autophagosome closurein occurred CK1?-depleted cells,indicating a novel role of CK1? in autophagosome closure.Then the effects of cell lines knocking down CK1? on ATG complex in the autophagy pathway were detected by the immunofluorescence,with the findings revealing that under starvation-induced autophagy in CK1?-depleted cells,the number of ATG16 L,ATG14L,and ATG9 A puncta structures showed no significant change,but the colocalization of LC3 with all three ATG proteins was increased,suggesting that knocking down CK1? can affect the dissociation of ATG proteins from autophagic membrane structure,which is consistent with the conclusions that CK1? can regulate autophagosome closure.To resolve the molecular mechanism of CK1? regulating autophagosome closure,CK1? interacting proteins were screened from proteins known to be involved in autophagosome closure with immunocoprecipitation to check the interaction between CK1? and reported regulators of autophagosome closure.We found that Syntaxin13(STX13),a SNARE protein involved in membrane fusion and autophagsome closure,interacted with CK1?.In summary,for the first time we found the essentail role of CK1? in regulating starvation-induced macroautophagy,specifically in regulating autophagosome closure,possibly by interacting with STX13,a SNARE protein involved in membrane fusion and autophagsome closure.This study not only reveal a novel role of CK1? in autophagosome closure,but also advance the molecular mechanism of autophagosome biogenesis.