Nek2A/SuFu Negative Feedback Loop Regulates Gli-mediated Hedgehog Signaling Pathway

Background:Hedgehog(Hh)signaling pathway was found by a genetic screen in Drosophila for mutants and played an important role in the development of organs of the body.More and more studies show that Hh signaling pathway is associated with the occurrence development of many tumors,such as basal cell carcinoma,non-small cell lung cancer,pancreatic cancerandbladder cancer.Transcription factors Gli is the terminal point of the cascade conduction in Hh,so it is also called the Hh/Gli signaling pathway.Suppressor of Fused(SuFu),one of the most conserved components of the Hedgehog(Hh)signaling,has an important function in Hh signaling.It has been reported that SuFu can inhibit the activity of Hh signaling pathway.Despite the central importance of SuFu in the Hh pathway,little is known about SuFu regulation.Nek2A is a member of the serine/threonine kinases Nek family and has an important role in the regulation of centrosome.There are mainly three types of Nek family proteins,namely Nek2A,Nek2B and Nek2 C.In vertebrates,the Nek family is expressed as two main splice variants Nek2A and Nek2 B.Nek2 has emerged as an important oncogene due to its regulatory role in mitosis and cancer-related signaling pathways.In tumor,high Nek2 expression showed poor prognosis.Our previous study found that,NIMA-related expressed kinase 2A(Nek2A)can be as a SuFu-interacting protein.Given the importance of SuFu and Nek2A in tumorigenesis,we further dissected the functional relationship of these two proteins.Hh/SuFu/Nek2A may provide a new strategy for human diseases.Objectives:1.Investigated the regulation mechanism of SuFu by Nek2A.2.Analyze the relationship of Nek2A and Hh signaling.3.Elucidate the molecular mechanism of NEK2A regulation by Gli1/2.Methods:1.The effect of Nek2A on the expression of SuFu and components of the Hh signaling pathway were performed by Western blot.2.The effect of Nek2A on SuFu stabilization was tested by immuneprecipitation.3.The influence of Nek2A on the mRNA of SuFu and components of the Hh signaling was detected by Real-time PCR.4.Molecular mechanism of Gli1/2 regulation NEK2A transcription was assayed by the Dual-Luciferase assay and ChIP.Results:1.Nek2A impairs ubiquitin/proteasome-mediated degradation of SuFu protein.2.Suppression of Nek2A activates Hh signaling pathway.3.Nek2A is regulated by Hh signaling.4.NEK2A is a target gene of transcription factor Gli.Conclusion:Gli1/2 directly activates NEK2A.Increased Nek2A protein then interacts with SuFu and promotes its stabilization.Stabilized SuFu subsequently inhibits Gli1/2 entering into the nucleus and suppresses Gli1/2 transcriptional function on NEK2A.