SUMOylation Of Large Tumor Suppressor 1 At Lys751 Attenuates Its Kinase Activity And Tumor-suppressor Functions

Large tumor suppressor(LATS)plays a critical role in maintaining cellular homeostasis and is the core to mediate Hippo growth-inhibitory signaling pathway by phorsphorylating YAP/TAZ directly.SUMOylation is a ubiquitin-like post-translational modification,it is a reversible and dynamic process that controls a variety of cell functions by regulating protein activity,stability,distribution and so on.In the present study,we demonstrated that SUMOylation of LATS1 affects its kinase activity and ultimately inhibits its tumor suppression function.Co-Immunoprecipitation was used to detect interaction between Small ubiquitin-like modifier(SUMO1)and LATS1,the results showed either endogenous or exogenous SUMO1 interacts with LATS1.At the same time,we also detected the co-localization of exogenous SUMOl and LATS1 by immunofluorescence,and we found SUMO1 and LATS 1 co-localized both in the cytoplasm and nucleus.In vitro SUMOylation assay were used to detect the SUMOyalated LATS1,which demonstrated that SUMO1 directly SUMOylates LATS1.SUMO1 G97A mutation or Ubc9 knock down,which lead to loss of SUMOylation pathway function,disrupted LATS 1 SUMOylation.We futher predicted the potential SUMOylation sites on LATS1 according to the motif characteristics of SUMOylation on specific website,and we got six sites including K49,K505,K622,K699,K751 and K830.All the lysine in the predicted sites were substituted by Arginine and Tead4-luciferase activity assay showed that the K751 and K830 are conversed and essential for maintaining the transcriptional output of Hippo signaling,K751R decreased the Tead transcription activity,while K830R mutation presented the opposite effect.Co-Immunoprecipitation was used to detect the amount of SUMOylation of K751R and K830R in 293T cell,the result showed that K751R mutation more significantly abolished SUMO 1-induced LATS1 SUMOylation than K830R mutation,demonstrating that K751 is the major SUMOylation site on LATS1.We further explored the effects of K751R mutation on Hippo signaling pathway.Firstly,we detected the protein expression of the core molecules in Hippo signaling pathway by Western blotting,and the results showed that K751R mutation upregulated the p-LATS1(T1079)expression and promoted the phosphorylation of downstream effector YAP/TAZ subsequently reduced the total YAP/TAZ expression.Secondly,western analyses were performed for YAP/TAZ in cytosolic versus nuclear fractions of HEK293T cells,and results shwed that the K751R mutation inhibited the nuclear translocation of YAP/TAZ,same phenomenon could be observed in the immunofluoresence study.Finally,the results of TEA domain transcription factors(Tead)-lnciferase activity assay proved that K751R inhibited Tead transcriptional,and the expression of target genes of Hippo signaling pathway was also detected by quantitative real time PCR,the results showed that K751R mutation inhibited the expression of Connective tissue growth factor(CTGF)and Cysteine rich protein 61(CYR61).Then we examined the mechanisms by which LATS1 SUMOylation at K751 regulating Hippo signaling.According the results of Immunofluorescence,we found LATS1 SUMOylation at K751 did not affect its subcellular distribution,and Co-Immunoprecipitation assay showed that the K751R mutation of LATS1 did not affect its interactions with YAP and TAZ.Then we studied the effects of K751R mutation on the degradation of LATS1 and p-LATS1 by using Cycloheximide(CHX),which was used to inhibit the synthesis of protein,the results proved that K751R mutation did not affect the stability of LATS1 but significantly stabilized the phosphorylated LATS1(Thr1079 but not Ser909).Moreover,we compared the amount of SUMOylated LATS1 in normal hepatic cells(L02)and in hepatocellular carcinoma cells(HepG2)by Co-Immunoprecipitation.HepG2 cells express significantly more SUMOylated LATS1 than L02 cells,and in HepG2 cells,LATS1 SUMOylation at K751 consistently attenuates LATS1 kinase activity and subsequently suppresses Hippo signaling,resulting in not only the promotion of cell proliferation and colony formation but also the suppression of cell apoptosis.Finally,human cancer cell HepG2 was either stably expressed with wild type LATS1 or K751R mutant,then these cells were subcutaneously inoculated into the nude mice to develop a xenograft model,and the volume of the tumor was measured regularly.We found the tumor growth rate of the K751R mutant group was significantly lower than that of the wild type LATS1.Futhermore,the expression of proliferation marker ki67 in the tumor tissue was detected by immunofluorescence,and the results showed that the expression of ki67 was significantly lower in K751R group than that in wild type LATS1 group.The TUNEL staining results of showed that the K751R mutation promoted the apoptosis of the tumor cells.Together,we demonstrate that LATS1 SUMOylation at K751 suppresses its kinase activity and subsequently attenuates its tumor-suppressor functions.Thus,this study provides additional insight into how Hippo signaling is regulated and highlights the potentially critical role of LATS1 SUMOylation in tumorigenesis.