Fig. 1

MEKK2/3 inhibit GLI1 transcriptional activity through their kinase activity. a MEKK2 and MEKK3 inhibited GLI1 transcriptional activity in HEK293T cells transfected with GliBS-luc reporter, GLI1, and indicated plasmids by a luciferase assay. b MEKK2 and MEKK3 inhibited Shh signaling activity in NIH3T3 cells transfected with GliBS-luc reporter and indicated plasmids by a luciferase assay. c MEKK3 inhibited expression of Hh signaling target genes Gli1 and Ptch1 in NIH3T3 cells transduced with MEKK3 lentivirus assayed by qRT-PCR analysis. d Endogenous MEKK2 and MEKK3 proteins were immunoprecipitated by GLI1-Flag proteins. Lysates from GLI1-Flag stable NIH3T3 cells were immunoprecipitated and immunoblotted as indicated. e Overexpression of MEKK3 induced a mobility shift of endogenous GLI1 in NIH3T3. Six percent SDS-PAGE was used to examine the mobility shift of GLI1. f MEKK2 and MEKK3 promoted phosphorylation of GLI1 in an in vitro kinase assay. MEKK2-Flag, MEKK3-Flag, and GLI1-HA proteins were synthesized using rabbit reticulocyte lysate system in vitro. Total phosphorylation of GLI1 was detected by immunoblotting with thiophosphate ester antibody, which identifies the alkylated thiophosphorylation on GLI1. g Alignment of identified phosphorylation sites in GLI1 by mass spectrometry across different species. Blanks indicate that there is no homolog sequence. Schematic representation of GLI1 molecule and phosphorylation sites (upper panel). Some GLI1 structural motifs, including SUFU binding site (SUFU-BS), zinc finger (ZnF), nuclear localization signal (NLS), nuclear export signal (NES), and transcriptional-activation domain (TAD), are denoted. h Expression of MEKK3 induced endogenous GLI1 phosphorylation in Hela, Daoy, and NIH3T3 cells. Cell lysates from lentiviral expression of MEKK3 were analyzed by western blot with indicated antibodies. i HEK293T cells transfected with GliBS-luc reporter and indicated plasmids were analyzed using a luciferase assay to measure GLI1-6A and GLI1-6D transcriptional activity. *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed Student’s t-test). All data were mean ± s.d. from representative of three independent experiments conducted in triplicate