Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors
Figure 7
Gli3 is a novel MAP kinase substrate with a functional D-site.
(A) Diagram of full length Gli3, shown with its transcriptional repressor domain (Rep Dom, purple rectangle), the Suppressor-of-Fused binding site (SuFu BS, green rectangle), and its 5 Zinc Finger (ZF) DNA-binding domains (gray oval). The position (triangle) and sequence of the D-finder-predicted D-site is also shown. Below, the Gli3280–478 wild-type (WT) and D-site mutant (DSM) fragments used for binding and kinase assays are shown, along with the sequence of the D-site mutant. (B) The Gli3280–478 wild-type and D-site mutant proteins were tested for binding to GST, GST-JNK1, GST-JNK2, GST-JNK3, and GST-ERK2 (40, 30, 30, 20 and 30 µg respectively). The upper two panels show the bound Gli3 derivatives, with 5% if the total input shown in lane 1; the lower panel shows Coomassie Blue (CB) staining of the sedimented GST-fusion proteins. Other details as in Fig. 4C. (C) Graph of the results of three independent repetitions of the binding assay shown in A and B, with duplicate points in each repetition. Standard error bars are shown (n = 3). (D) In vitro kinase assays assessing the phosphorylation of the WT and DSM fragments of Gli3 by active JNK1 and JNK2. Three separate concentrations of substrate (0.1, 0.3 and 0.5 µM) were incubated with 0.5 mU (∼1 ng) of active enzyme. Image is representative of three independent experiments. Other details as in Fig. 4E. (E) In vitro kinase assay assessing the phosphorylation of the WT and DSM fragments of Gli3 by activated ERK2. Substrate concentration: 0.5 µM. Enzyme activity: ERK2 – 0, 1, or 10 units (10 units is ∼1 ng).