Figure 1.
MAP kinases interact with D-sites on substrates and regulators.
(A) JNK and several classes of JNK-interacting proteins. The D-site on JNK-binding proteins is shown as a triangle. (B) Literature-verified JNK D-sites that were used as a training set for the hidden Markov model component of D-finder. (C) Sequence logo [111] of the central 14 residues of the D-sites shown in B.
Figure 2.
Validation of the D-learner hidden Markov model.
(A–C). The HMM accurately identifies known D-sites in full-length sequences. Full-length sequences run through the HMM give a Viterbi probability for every window tested. The x-axis displays the window number and the y-axis shows the log of the Viterbi probability for each window. The dashed lines represent the threshold of E-23 for a window to be considered a predicted D-site. MKK4 (A) has one peak, MKK7 (B) has three peaks, and the arbitrarily-chosen full length sequence SEMA3C (C) has zero peaks above the threshold. (D) The HMM does not score randomized sequences highly, even if they have the same composition as a high-scoring D-site. Histogram of scores assigned to 1,000 scrambled sequences with same sequence composition as the MKK4 D-site (blue, left ordinate labels) and the 20 training set D-site sequences (green, right ordinate labels). Sequences were binned by score, with no sequences scoring below −37 or above −14. For the MKK4 randomized set, zero sequences surpassed the −23 threshold (dashed line). For the 20,000 total randomized D-site sequences, 30 sequences (0.15%) scored above this threshold. For the training set, 16 sequences (80%) surpassed the E-23 threshold. (E) The HMM scores JNK D-sites higher than D-sites selective for ERK- or p38-family MAPKs. The name, D-learner-assigned score, and sequence of all known human MKK D-sites are shown. The JNK D-sites (MKK4 and the 3 MKK7 D-sites) surpass the −23 threshold; however, the non-cognate D-sites, although they contain the core consensus basic (blue) and hydrophobic (red) residues, do not score above the threshold.
Figure 3.
D-finder architecture and results of human genome search.
(A) Overview of D-finder. D-finder consists of D-matcher, a pattern matching algorithm employing expert knowledge, and D-learner.T1, a profile HMM trained on the training set shown in Fig. 1B. D-matcher filters out most windows, but found many acceptable windows in most sequences. D-learner assigns a probability score to each window it is passed, and found above-threshold windows in only 403 of the sequences passed to it by D-matcher. When D-learner was run without D-matcher interceding, it found 2,260 above-threshold windows in 1,784 sequences. (B) The top 25 D-sites found by D-finder in the human genome.
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.
Figure 5.
Identification of a D-site in the PPM1J phosphatase.
(A) Wild-type (WT) and N-terminal truncated versions (NT w/o D) of PPM1J were fused to GST and tested for binding to JNK 1–3. The sequence of the D-site is shown. (B) As shown in A, 35S-radiolabeled JNK1, 2 or 3 (∼1 pmole) were tested for binding to 40 µg of GST (lane 1) or 30 µg of GST-PPMIJ K17–506 (containing the D-site, lanes 3 and 4) or GST-PPMIJ K80–506 (lacking the D-site, lanes 5 and 6). Lane 1 shows a 5% of the total JNK input. The lower panel shows Coomassie Blue (CB) staining of the sedimented GST-fusion proteins. Other details as in Fig. 4C. (C) Wild-type (WT) and D-site-mutant (DSM) versions of PPM1J were tested as substrates for in vitro phosphorylation by active JNK1-3. (D) As shown in C, 1 µM of each GST-PPM1J protein was incubated with 0, 0.1 or 1 mU JNK1-3 and [γ-32]ATP for 20 min. Incorporation of radioactive phosphate into the substrate, as assessed by autoradiography, is shown in 3 panels, and a representative Coomassie blue (CB) stained gel, to demonstrate equal loading of the substrates proteins, is shown. (E) Wild-type and D-site mutant versions of PPM1J were C-terminally-tagged with the V5 epitope, expressed in Cos-1 cells, immunoprecipitated, and used as substrates for JNK1-mediated phosphorylation. (F). As shown in E, Cos-1 cells were transfected with either empty vecor (EV), PPM1J-V5 WT, or PPM1J-V5 DSM. 16 h post-transfection, the cells were harvested, lysed, and immunoprecipitated with anti-V5 antibodies. MAPK buffer, [γ-32]ATP, and active JNK1 were added to the immunoprecipitated pellets, and phosphorylation of the immunoprecipitated proteins was visualized by PhosphorImager. In addition, portions (20 µg) of each lysate were separated by SDS-PAGE and immunoblotted (WB, for Western Blot) with either anti-V5 (1∶5000) or anti-total JNK (1∶500) antibodies.
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.
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).
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.
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.