Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors
Figure 4
Identification of a D-site in the known JNK substrate hnRNP-K.
(A) Full-length hnRNP-K protein. KH, K homology domain; KI, K interaction domain. The positions of known JNK and ERK phosphosites and the D-finder-predicted D-site are shown, with key consensus basic (blue) and hydrophobic (red) residues highlighted by color. (B) Wild-type (WT) and D-site mutant versions (DSM) of hnRNP-K were tested for binding to GST-JNK1. The sequence of the D-site mutant is shown. (C) As shown in B, 35S-radiolabeled full-length hnRNP-K protein and a D-site mutant derivative were prepared by in vitro translation and partially purified by ammonium sulfate precipitation, and portions (5% of the amount added in the binding reactions) were resolved on a 10% SDS-polyacrylamide gel (lane 1). Samples (∼1 pmol) of the same proteins were incubated with 40 µg of GST (lane 2) or with 10 to 40 µg of GST-JNK1 (lanes 3–6), bound to glutathione-Sepharose beads, and the resulting bead-bound protein complexes were isolated by sedimentation and resolved by 10% SDS-PAGE on the same gel. The gel was analyzed by staining with Coomassie Blue (CB) for visualization of the bound GST fusion protein (a representative example is shown in the lowest panel) and by Phosphorimager analysis for visualization of the bound radiolabeled protein (upper two panels). (D) Fragments of hnRNP-K were tested as substrates for in vitro phosphorylation by active JNK. (E) As shown in D, GST fusions to hnRNP-K281–464 (containing the D-site, w/D) and hnRNP-K317–464 (deleted of the D-site, w/o D) were purified and incubated with purified active JNK1 and [γ-32]ATP for 20 min. Substrate concentration: 500 nM; Enzyme activity: 0, 0.8 mU, or 8mU. Reaction products were separated by SDS-PAGE and incorporation of radioactive phosphate into the substrate was assessed on a PhosphorImager.