SUMOylation Is Required For PIPK1γ-driven Keratinocyte Migration And Growth

ObjectiveThe aim of this study was to explore the function of PIP5K1 and its SUMOylation modification on keratinocyte.MethodsPart one: PIP5K1γ can be SUMOylated.HA-PIP5K1α,HA-PIP5K1β and HA-PIP5K1γ were co-transfected into HEK293 T.Co-immunoprecipitation(IP)assay was used to analysis whether type I PIPKs could associate with UBC9;HA-PIP5K1γ、FLAG-SUMO1 with(or without)Myc-UBC9 were then co-transfected into HEK293 T to find whether PIP5K1γ could be SUMOylated through IP and Western Blot.HA-PIP5K1γ,FLAG-SUMO1,RGS-HIS-SENP1 and RGS-HIS-SENP1 mutant was transfected into HEK293 T to confirm the SUMOylation of PIP5KIγ through immunoprecipitation and western blot.Then,PIP5K1γ,GST-PIP5K1γ and pE1E2S1 were transfected into E.coli,after co-culture,the protein was extracted from the bacteria,and the SUMOylation of PIP5K1γ was detected by Western blot.Endogenous PIP5K1γ was also found to be modified by endogenous in keratinocytes HaCat by immunoprecipitation and western blot.HA-PIP5K1γ-WT,HA-PIP5K1γ-KD and FLAG-SUMO1 were transfected into HEK293 cells and then to assess immunoprecipitation was performed to make sure whether the SUMOylation of PIP5KIγ is dependent on its kinase activity.Part two: To identify the SUMOylation sites of PIP5K1γ and explore the influence of PIP5K1γ on the proliferation and migration of HaCat.Firstly,the potential SUMOylation sites of PIP5K1γ were predicted through bioinformatics.The wild-type of PIP5K1γ(PIP5K1γ-WT),and 3 mutants of PIP5K1γ(PIP5K1γ-K251 R,PIP5K1γ-K408 R,PIP5K1γ-K591R)and FLAG-SUMO1 were co-transfected into HEK293 T to test whether these 3 Lys are SUMO-acceptor sites.PIP5K1γ-WT,PIP5K1γ-K591 R and pE1E2S1 were then co-transfected into E.coli to further confirmed the SUMOylation sites of PIP5K1γ.PIP5K1γ was knocked out in HaCat using CRISPR/CAS9 technology.The cell viability of HaCat cells was detected by using IncuCyte ○R S3 Live-Cell Analysis System.The migration of HaCat cells was observed by wound healing experiment.Western blot was used to detect the classic biosensors involved in tumor cell proliferation and migration.Part three: To clarify the significance of SUMOylation in PIP5K1γ-driven keratinocyte migration and growth,PIP5K1γ-WT and SUMOylation-deficiency mutant were reconstructed on the basis of PIP5K1γ-null HaCat.The migration ability of HaCat cell was observed by the scratch assay and was further clarified by Transwell assay.Meanwhile,the cell proliferation capacity was detected by using IncuCyte? S3Live-Cell Analysis System.Cytoplasm,cell membrane and nuclear protein were separated by plasma membrane separation kit to verify whether SUMOylation is necessary for the location of PIP5K1γ in cells.The proteasome inhibitor MG132 was used to observe whether SUMOylation could affect the protein stability of PIP5K1γ.CHX was used to observe whether SUMOylation could affect the protein half-life of PIP5K1γ.HA-PIP5K1γ-WT,SUMOylation-deficiency mutant K591 R and HIS-ubiquitin were transfected into HEK293 T to observe whether SUMOylation affected the ubiquitination of PIP5K1γ.PIP2 was synthesized from PIP5K1γ-null,PIP5K1γ-wt and PIP5K1γ-K591 R and then detected by Elisa.Conclusion:1.PIP5K1γ can be SUMOylated in vivo and vitro.2.The expression of PIP5K1γ affected the proliferation and migration of keratinocyte.3.The SUMOylation of PIP5K1γ affected the proliferation and migration of keratinocyte through the generation of PIP2.