Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors

Abstract

In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates.

Two Hydrophobic Residues Can Determine the Specificity of Mitogen-activated Protein Kinase Docking Interactions

“…Conclusions-We have investigated the sequence features of MAPK-docking sites that influence their ability to bind to cognate, within-pathway MAPKs over non-cognate MAPKs. Our findings provide insight into specificity in MAPK signaling networks and should be relevant to efforts to develop docking inhibitors (13)(14)(15)(16)(17) and to predict new MAPK substrates and regulators (75). …”

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…Indeed, Ser/Thr phosphatases can directly regulate JNK actions. For example, the protein Ser/Thr metallophosphatase PPM1J (PP2C) was found to harbor a JNK-binding motif (69). Since this recruitment or docking motif was also found in the related PPM1H phosphatase and is broadly conserved across the animal kingdom, these phosphatases may have important roles in the regulation of JNK pathways.…”

“…In recent years, many novel JNK partners (mostly substrates) have been identified based on the presence of D-motifs, and their number is expected to grow (69). JNK-associating D-motifs can be separated into at least two, structurally different varieties: either resembling the D-motif found in the JNK pathway regulator JIP1 or resembling the motif described for the NFAT4 transcription factor (80,200) (Fig.…”

“…Two phosphorylation sites of the recently identified JNK substrate SMTL2 (a close relative of the actin-binding protein smoothelin) also lie in a conserved motif resembling a near-optimal FBW7 phosphodegron (PLVTPPQSPVS; phosphorylation sites are underlined). Although the functions of SMTL2 require further exploration (69), these examples provide interesting insights into proteins regulated through the interplay of JNK-mediated phosphorylation and their ensuing degradation.…”

“…Cross talk of JNK with two other developmentally important signaling pathways (Hedgehog and Hippo) has also been suggested. Gli is an important terminal regulator of Hedgehog signaling, and its JNKmediated phosphorylation suggests that a direct connection between MAPK and Hedgehog signaling may occur at the level of these key regulators (69). Recently, JNK-mediated inhibition of Hippo signaling was described as a mechanism allowing mechanical strain to influence cell proliferation (286).…”