Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases

Abstract

Mitogen‐activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less‐characterized disordered regions. We used a structurally consistent model on kinase‐docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under‐explored part of the human proteome and applied experimental tools specifically tailored to detect low‐affinity protein–protein interactions for their validation in vitro and in cell‐based assays. The combined computational and experimental approach enabled the identification of many novel MAPK‐docking motifs that were elusive for other large‐scale protein–protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase‐mediated partnerships evolved over time. The analysis suggests that most human MAPK‐binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK‐binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.

Defining Human Tyrosine Kinase Phosphorylation Networks Using Yeast as an In Vivo Model Substrate

“…Cantley and coworkers recently reported crystal structures of the EGFR kinase domain with a bound peptide substrate where the peptide residues did not have well-defined electron density despite the use of an optimized linear sequence and concluded from their structural observations that, other than the +1 residue, the primary sequence surrounding the phosphorylation site may have little influence on EGFR specificity (Begley et al, 2015). Other work has shown involvement of docking sites, targeting subunits and scaffolds, which better explain kinase-substrate specificity through additional protein interactions (Bhattacharyya et al, 2006;Ubersax and Ferrell, 2007;Zeke et al, 2015). However, since those additional protein interactions may not all exist in the yeast model substrate, the physical and topological constraints on substrates in the cell could also contribute.…”

“…Therefore kinase specificity determinants were mainly assayed with purified kinases and synthetic peptide substrates in vitro. As a result, while specificity determinants such as docking sites have been reported (Ubersax and Ferrell, 2007;Zeke et al, 2015), the primary amino acid sequence surrounding the phosphorylation site, referred to as kinase motif, is the predominant specificity determinant studied to date (Miller et al, 2008;Mok et al, 2010;Deng et al, 2014;Duarte et al, 2014).…”

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

“…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. 5; see also Table 1 for a summary of D-motif types in known JNK substrates).…”

“…The role of the rhodanese domains in MKP1 and MKP5 in recruiting ERK2 and p38␣ is well-known: by mimicking a so-called docking motif, this surface enables strong interactions with several MAPKs (79). However, the docking site of JNK cannot bind rhodanese domains, so phosphatases targeting JNK bind in alternative ways (via their catalytic domain or by docking motifs, as observed in MKP5 and MKP7 [80,81]). In vivo studies suggest that MKP1 is not required for growth factor signaling; however, it is essential for immune cell activation, as MKP1 Ϫ/Ϫ mice show immune defects (82).…”

“…Despite these alternative modes of interactions with the MAPKs, most JNK interactors use typical D-motifs to partner with JNKs (80).…”