Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors
Figure 6
Testing predicted D-sites with peptide arrays.
(A) Membrane-attached peptides were probed for binding to radioactively-labeled JNK1. Peptides containing functional JNK-docking sites (e.g. the MKK4 or MKK7-D2 positive controls or accurate predictions) bound to JNK1, while those containing non-binding peptides (e.g. negative controls or false predicitions) did not. (B) Representative examples of peptide arrays probed with 35S-labeled JNK1 and then visualized and quantified by Phosphorimager. Controls (circled, in duplicate on each membrane) are the published D-sites of MKK4 and MKK7-D2, and their mutants with alanine substitutions at the critical basic and hydrophobic residues. The binding efficiency of the average of the training set peptides and the positive and negative controls are plotted; this has been normalized by setting the efficiency of the MKK7-D2 positive control to 100%. (C) Plot of the normalized binding percentages (with S.E.M. bars) for the 59 predicted D-site peptides that were tested. The threshold for classification as positive is 100%. Red-colored bars are above threshold, green-colored bars are below threshold.