Ation of your BCAR4 RNA probe (nt 235-288) and (nt 991-1044) with recombinant SNIP1 and PNUTS, respectively, resulted in particular gel retardation (Figure 2H). Below these circumstances, no shift was observed when the corresponding cold probes have been utilised (Figure 2H). We, consequently, conclude that BCAR4 straight bind to SNIP1 and PNUTS by means of two distinct regions. Given MS mAChR4 Gene ID information displaying that GLI2 is phosphorylated at Ser149 and associates with CIT kinase (see Figures 2A and S2B), we reasoned that CIT might serve as a kinase to phosphorylate GLI2. In vitro kinase assay indicated that bacterially-expressed wild form GLI2 was phosphorylated by CIT, but not S149A mutant (Figure S2F). ULK3 served as the positive control because of its reported capability to phosphorylate GLI (Maloverjan et al., 2010). In vitro RNA-protein binding assay working with biotinylated BCAR4 and GLI2 proteins phosphorylated by CIT in vitro showed no interaction (Figure S2G).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCell. Author manuscript; out there in PMC 2015 November 20.Xing et al.PageTo investigate the function of GLI2 Ser149 DYRK4 Source phosphorylation in vivo, we generated rabbit polyclonal antibodies that especially recognized Ser149-phosphorylated GLI2 known as p-GLI2 (Ser149) antibody, which specifically detected bacterially-purified GLI2 protein that phosphorylated by CIT in vitro, with minimal reactivity towards GLI2 phosphorylated by ULK3 (Figure 2I). We conclude that p-GLI2 (Ser149) antibody specifically recognizes CIT-mediated Ser149 phosphorylation of GLI2. Next, we evaluate the amount of phosphoGLI2 in breast cancer by immunohistochemistry (IHC) analysis of clinical tumor specimens, acquiring greater p-GLI2 (Ser149) levels in invasive breast cancer tissues compared with adjacent regular tissues (p=0.0087) (Figure 2J). Our IHC staining additional revealed increased p-GLI2 (Ser149) level in several cancer forms compared to their corresponding regular tissues (Figure S2H; Table S5). IHC evaluation also revealed larger CIT expression in invasive breast cancer compared with adjacent normal breast tissues (p=0.0055) (Figure S2I) plus the staining of phosphorylated GLI2 strongly correlated with that of BCAR4 and CIT staining (Information not shown). Taken together, we identified and characterized that BCAR4 binds a protein complicated containing SNIP1, PNUTS, phosphorylated GLI2 and CIT through its direct interaction with SNIP1 and PNUTS. CCL21 Induces GLI2 Ser149 Phosphorylation and Nuclear Translocation of Phosphorylated GLI2 The CIT kinase-mediated GLI2 phosphorylation prompted us to investigate whether this phosphorylation may be triggered in MDA-MB-231 cells by hedgehog signaling. Surprisingly, although the ligand SHH activated hedgehog signaling in Daoy cells evidenced by stimulated SHH gene induction as previously reported (Wang et al., 2012), minimal impact was observed in MDA-MB-231 cells (Figure S3A) and no phosphorylated GLI2 was detected (information not shown), suggesting that a noncanonical hedgehog signaling pathway, involving Ser149-phosphorylated GLI2, could exist in breast cancer. We then explored irrespective of whether extracellular signals that activate CIT kinase could also trigger GLI2 phosphorylation in breast cancer cells. Given that CIT kinase can be activated by GTPase Rho proteins (Madaule et al., 1998), we initial screened the CIT-Rho interaction in breast cancer cells. Even though CIT kinase is constitutively linked with RhoA as previously reported (Gai et al., 2011), the presence.