Study On The Interaction And Molecular Mechanism Of Nek2A And Sufu

Hedgehog signaling pathway plays an important role in cell growth,embryogenesis and maintaining homeostasis.It is also reported to be involved in tumorigenesis and tumor progression,such as basal cell carcinoma,gastrointestinal cancer,ovarian cancer and so on.A major member of Hedgehog signaling pathway is consist of Hh ligand,Ptch of 12 transmembrane protein receptor,SMO of 7transmembrane protein,Sufu of cytoplasmic negative regulatory protein,transcription factor Gli and downstream related target genes.Sufu?Suppressor of fused?as an important regulator of the Hedgehog signaling pathway,mainly acts as an inhibitor of Hedgehog signaling through impairing the activity of downstream transcription factor Gli,but little know about Sufu regulation.In order to figure out Sufu protein regulation,we use yeast two hybrid method by Sufu as bait and obtain unknown interactions of protein,including Nek2?NIMA-related kinase 2?.Nek2,a cell cycle related serine/threonine kinase,high expression in the proximal end of the replicated 2 centrosome.However,about 90%Nek2 of those asynchronous cells located outside the centrioles and may have other unknown function.In mammalian cells,Nek2 has three isoforms,which are Nek2A,Nek2B,Nek2C,and Nek2A is the full length form.Nek2A is highly expressed in a variety of tumor tissues,and is highly expressed in patients with poor prognosis.Therefore,it is important to study the interaction and molecular mechanism of Nek2A and Sufu protein.It can enrich the regulation network of Hedgehog signaling pathway,which is of great significance to reveal the development of tumor.Methods:1.Interaction between Nek2A and Sufu and binding domain assay.The interaction between two proteins was verified by the co-immunoprecipitation of exogenous and endogenous proteins.Binding domain was found by GST pull down technique.2.The protein level of Sufu by Nek2A affecting and the molecular mechanism of interaction.Through overexpression and interference method,the protein level change of Sufu was detected by Western blot and mRNA level change of Sufu was detected by Q-PCR.phosphorylation level was detected by specific phosphorylation of antibody.phosphorylation sites analyzed by mass spectrometry.3.Nek2A affects the activity of Hedgehog signaling pathway through interaction with Sufu.Luciferase activity was detected by using the dual luciferase reporter assay system.Protein level of transcription factor Gli2 in cytoplasmic or nuclear was detected by subcellular fractionation.4.the ciliary localization of Sufu was analyzed by the cellular immunofluorescence.Results:1.We discovered the interaction between Sufu and Nek2A through yeast two-hybrid experiment,and proved that Nek2A interacted with Sufu in mammalian cells through exogenous and endogenous co-immunoprecipitation.Then we further identify the binding domain of Sufu with Nek2A through GST pull–down assay,and eventually confirmed that Sufu?1-367 aa?is the interaction site.2.In cells overexpressed Nek2A,we found that the protein level of Sufu was significantly elevated,while the mRNA level did not change significantly,indicating that Nek2A does not regulate the synthesis of Sufu,but the degradation of it.Besides,the degradation of Sufu protein was slowed down in the experimental group under the treatment of cycloheximide for inhibition of protein synthesis.We also found that the SuFu protein level was reduced when Nek2A kinase dead mutant over-expressed,suggesting that effect of Nek2A on stabilizing Sufu protein requires Nek2A kinase activity.We found that the phosphorylation level of Sufu protein was enhanced in the Nek2A over-expressed cells by the detecting with phosphor-serine/threonine antibody.Furthermore,using mass spectrometry analysis technology,we found that two sites of Sufu protein?T225 and S352?were specifically phosphorylated by Nek2A.In order to verify these phosphorylation sites,we constructed Sufu point mutation plasmid(Sufu^T225/S352A),and found that the mutant possessed a decreased protein stability when compared with wild-type Sufu.3.We used dual luciferase reporter assay system to detect the transcription activity of Gli2.After transfected with Nek2A,the fluorescent signal value decreased,suggesting that the inhibition of Hedgehog signaling pathway by Nek2A is probably due to stabilizing Sufu.After subcellular fractionation,we found that the nuclear content of Gli2 decreased in Nek2A overexpressed cells.4.We labeled cilia by acetylated tubulin antibody and found that the ciliary localization of Sufu was increased?P<0.05?when Nek2A was knocked down transiently.It further indicates that Nek2A can negatively regulate the ciliary hedgehog signaling.Conclusion:1.Nek2A interacts with the Sufu protein,and binds to the N side of the Sufu.2.Nek2A can stabilize the Sufu protein depending on its kinase activity.3.After stabilized by Nek2A,Sufu protein has the function of inhibiting the activity of Hedgehog signaling pathway.