The Role Of MEKK2/3 In Regulating Hedgehog Signaling Pathway

MEKK2/3 are serine/threonine kinases coded by Map3k2/3 gene from MAP3K(mitogen-activated protein kinase kinase kinase)family.There is high homology of amino acid sequence in the kinase domain of MEKK2 and MEKK3.Thus,these two kinases are always studied together in plenty of articles involved in kinase regulation.As the vital initiation of MAPK cascades,the activation of MEKK2/3 is regulated by extracellular signals and stresses,signals including the growth factors,such as FGF2(fibroblast growth factor 2),EGF(epidermal growth factor)and inflammatory cytokines,such as TNFa(tumor necrosis factor a),IL-1?(interleukin-1?);stresses including osmotic stress,ultraviolet radiation and heat shock.Hedgehog signaling regulates cell proliferation,differentiation and organ formation,which plays a crucial role in many aspects of embryonic development.In postnatal physiology,Hedgehog signaling pathway is also important in stem/progenitor cells regulation,tissue homeostasis and regeneration,cancer development.GLI1(glioma-associated oncogene 1)is the component as well as target gene of Hedgehog signaling pathway,and the aberrant activation of GLI1 is associated with various types of human cancers,including basal cell carcinoma,medulloblastoma,breast cancer,etc.Therefore,the discovery of the critical factors that regulate GLI1 or Hedgehog signaling pathway will provide a certain theoretical significance to clinical therapies against Hedgehog pathway-dependent carcinoma.We performed screening on almost 500 kinases through GLI1 luciferase reporter assay.We found that MEKK2 and MEKK3 remarkably suppress GLI1 transcriptional activity and functionally inhibit the cell growth of medulloblastoma Daoy cells.In tumor formation assay,we subcutaneously injected the MEKK3-overexpressed Daoy cells and Mekk2/3-double-knockout Daoy cells respectively into nude mice.The results showed that the tumor growth rate of the latter one is much higher than that of the former one.Mechanically,through co-immunoprecipitation assay,kinase assay in vitro and mass spectrometry,we found that MEKK2/3 interact with GLI1 and directly phosphorylate it at multiple sites.Furthermore,MEKK2/3 promote the interaction between GLI1 and ?-TrCP,which accelerates the degradation of GLI1 by proteasome.The results of co-immunoprecipitation assay,immunofluorescence assay,etc showed that MEKK2/3 increase the association of GLI1 with SUFU(suppressor of fused),which leads to the cytoplasmic retention of GLI1.We also found that MEKK2/3 reduce the DNA-binding ability of GLI1 via DNA pull-down assay in vitro.Previous studies reported that FGF2 inhibits hedgehog signaling in neuronal precursors and tumor cells,but the inhibitory mechanism was not well elucidated.We found that FGF2 can activate MEKK2/3 to inhibit the expression of Hedgehog signaling pathway target genes.Utilizing the ChIP(chromatin immunoprecipitation)assay,we found that GLI1 bindsu to a conserved motif localized in the promoter of MEKK3 to promote the transcription of MEKK3.These results reveal a novel feedback regulation on Hedgehog signaling pathway.That is MEKK2 and MEKK3 as target genes of Hedgehog signaling to suppress GLI1 transcriptional activity through a feedback loop.Our study deciphers the mechanisms underlying FGF2-induced suppression on Hedgehog signaling,and provides a theoretical basis for therapeutics against Hedgehog signaling-dependent carcinoma.