Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors
Figure 9
Gli1 is a novel MAP kinase substrate with a functional D-site.
(A) Sequence alignment of Gli1 and 2 with Gli3 in the regions around the validated D-site (282–301 in Gli3) and target phosphosite (Ser 343 in Gli3). (B) Diagram of full length Gli1, shown with its 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 Gli168–232 wild-type (WT) and D-site mutant (DSM) fragments used for binding and kinase assays are shown. (C) The Gli168–232 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 Gli1 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. (D) In vitro kinase assays assessing the phosphorylation of the WT and DSM fragments of Gli1 by active JNK1, JNK2, JNK3 and ERK2. Three separate concentrations of substrate (0.1, 0.3 and 0.5 µM) were incubated with the indicated units of active enzyme. Image is representative of three independent experiments. Other details as in Fig. 4E. (E) As in D, but with the addition of the S130A mutant of Gli1.