Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors
Figure 8
D-site-directed phosphorylation of Gli3 Ser343.
(A) There are five putative MAPK target sites in the portion of Gli3 protein found to be phosphorylated in this work. To determine which sites were phosphorylated, Gli3 was incubated with active JNK2. (B), (C) & (D). Mass spectrometry analysis of an identified phosphorylated peptide (m/z 851.40, GHLSASAIS(phospho)PALSFTY). Four samples were analyzed: WT Gli3 with active kinase, DSM with active kinase, WT with no kinase, and DSM with no kinase. (B) MS/MS spectra of the identified peptide in the GST-GLI3280–478 WT plus active kinase sample. bi* = [bi−H3PO4]; yi* = [yi−H3PO4]. (C) Extracted ion chromatograms (XIC) of the parent ion from the four samples during LC MS runs. (D) MS of parent ion. (E) Results of an in vitro kinase assay assessing the phosphorylation of the WT, S343A, and DSM fragments of GLI3280–478 by activated JNK1, JNK2 and JNK3. Image is representative of three separate trials. The Coomassie Blue stained panels demonstrate equal loading of substrates. Substrate concentration: 1 µM. Enzyme activity: 0, 0.2, or 1 mU.