Substrate Discrimination among Mitogen-activated Protein Kinases through Distinct Docking Sequence Motifs

Abstract

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms.Mitogen-activated protein kinases (MAPKs) 4 lie at the bottom of conserved three-component phosphorylation cascades that integrate cellular responses to a wide variety of extracellular stimuli, including growth factors, cytokines, UV irradiation, and oxidative stress (1). Canonical MAPKs are classified into three major subfamilies, extracellular signal-regulated kinases (ERK), p38 MAPK (p38), and c-Jun N-terminal kinases (JNK), based on sequence homology, shared upstream kinases, and activating stimuli. In addition, the different MAPK subfamilies phosphorylate a distinct set of protein substrates. These substrates act as the critical effectors that enable cells to mount the appropriate responses to varied stimuli.Analysis of reported MAPK substrate phosphorylation sites suggests a nearly absolute requirement for a Pro residue immediately downstream of the Ser or Thr phosphoacceptor. Extensive peptide library studies on ERK1, ERK2, and p38␣ have defined a common phosphorylation site motif for these MAPKs. In addition to the requirement for a Pro residue at the ϩ1 position relative to the phosphorylation site, this motif includes a weak preference for Pro and other aliphatic residues at the Ϫ2 position (2-4). Mutagenesis experiments with the JNK substrate JunB have also suggested additional specificity for JNK family MAPKs at positions downstream of the phosphorylation site (5). MAPKs have affinities for short peptide substrates that are at least an order of magnitude lower than for full-length protein substrates (4, 6 -10). Thus although phosphorylation site motifs likely serve to direct the MAPK to phosphorylate a specific serine or threonine residue within a protein, they are insufficient to fully account for protein substrate recognition. Although mechanisms such as subcellular localization and the use of scaffolding proteins can contribute to kinase substrate targeting in vivo, MAPKs also exhibit a high degree of target specificity in vitro (11-13).For MAPKs, substrate specificity is ensured through the use of docking...

p38αMAPK interacts with and inhibits RARα: suppression of the kinase enhances the therapeutic activity of retinoids in acute myeloid leukemia cells

“…Identical phenomena were observed after mutation of Tyr-258 (Y258A), a residue stabilizing the structure of the interaction pocket responsible for the binding of p38a to another docking site (DEF domain) present on substrate proteins. 56 In contrast, two further mutations (I116A and Q120A) outside the canonicaldocking sites exerted no effect on p38a ability to interact with RARa. This correlated with the inability of mCD, mED and Y258A to inhibit ligand-dependent transactivation of RARa as well as PML-RARa (Supplementary Figure 5).…”

Mycobacterium tuberculosis Mce3E Suppresses Host Innate Immune Responses by Targeting ERK1/2 Signaling

“…Several effectors from pathogenic bacteria such as Legionella effectors display distinctive eukaryotic domains, among which are protein kinase or phosphatase domains (33)(34)(35). In our efforts to search for the functional domains/ motifs within Mce3E, we identified a potential DEF motif, an ERKdocking motif that possesses a consensus sequence of FXFP, in Mce3E (FPFP) using in silico analysis (http://scansite.mit.edu/) (36). Because the former Phe residue is most critical among the three conserved amino acids of the DEF motif, we thus mutated the Mce3E Phe294 into Ala (F294A), and found that this mutant could barely bind to ERK in vitro (Fig.…”

Opposing effects of Elk-1 multisite phosphorylation shape its response to ERK activation

“…Thus, the ERK docking motifs differentially affect each phosphorylation site's competitive behavior. Previous studies showed that Elk-1 TAD phosphorylation by JNK and p38 MAP kinase differs from phosphorylation by ERK (10,(21)(22)(23)(24), and that this reflects differences in their docking interactions (12,14,15,25). Indeed, these kinases exhibited site preferences and phosphorylation rates that were distinct from that of ERK2 ( fig.…”