Local destabilization, rigid body, and fuzzy docking facilitate the phosphorylation of the transcription factor Ets-1 by the mitogen-activated protein kinase ERK2

Abstract

Mitogen-activated protein (MAP) kinase substrates are believed to require consensus docking motifs (D-site, F-site) to engage and facilitate efficient site-specific phosphorylation at specific serine/ threonine-proline sequences by their cognate kinases. In contrast to other MAP kinase substrates, the transcription factor Ets-1 has no canonical docking motifs, yet it is efficiently phosphorylated by the MAP kinase ERK2 at a consensus threonine site (T38). Using NMR methodology, we demonstrate that this phosphorylation is enabled by a unique bipartite mode of ERK2 engagement by Ets-1 and involves two suboptimal noncanonical docking interactions instead of a single canonical docking motif. The N terminus of Ets-1 interacts with a part of the ERK2 D-recruitment site that normally accommodates the hydrophobic sidechains of a canonical D-site, retaining a significant degree of disorder in its ERK2-bound state. In contrast, the C-terminal region of Ets-1, including its Pointed (PNT) domain, engages in a largely rigid body interaction with a section of the ERK2 F-recruitment site through a binding mode that deviates significantly from that of a canonical F-site. This latter interaction is notable for the destabilization of a flexible helix that bridges the phospho-acceptor site to the rigid PNT domain. These two spatially distinct, individually weak docking interactions facilitate the highly specific recognition of ERK2 by Ets-1, and enable the optimal localization of its dynamic phospho-acceptor T38 at the kinase active site to enable efficient phosphorylation.MAP kinase | transcription factor | proximity-mediated catalysis | solution NMR T he mitogen activated protein (MAP) kinase ERK2 (extracellular signal-regulated kinase 2) lies at the terminus of a three-tiered phosphorylation-based response to a wide range of extracellular cues that include cytokines, hormones, and growth factors (1-4). ERK2 phosphorylates numerous substrates in both the nucleus and the cytoplasm, including a variety of transcription factors, regulatory kinases, phosphatases, proteins of the nuclear pore complex, and cytoskeleton proteins, among others (3, 4). ERK2-mediated phosphorylation occurs on specific serine/threonine residues that immediately precede a proline; however, the (S/T)P motif alone does not provide sufficient substrate affinity or selectivity to distinguish it from other proline-directed kinases, such as CDK2 (5). To attain high specificity toward native substrates, ERK2 and other MAP kinases use one of two so-called "docking sites," the D-recruitment site (DRS) or the F-recruitment site (FRS) (SI Appendix, Fig.

Extracellular Signal-Regulated Kinase: A Regulator of Cell Growth, Inflammation, Chondrocyte and Bone Cell Receptor-Mediated Gene Expression

“…In addition to the important role played by DUSPs in regulating overall ERK activity, activated ERK1/2 catalyzes the phosphorylation of numerous nuclear transcription factors, exemplified by ETS; ELK-1, a member of the ETS oncogene superfamily) [46,47]; c-Fos, a component of the activator protein-1 (AP-1) complex [48]; and Forkhead Box O (FOXO) [49], all of which arise through both active and passive nuclear translocation of phosphorylated ERK1/2 [1,50]. Of note, constitutively activated RAS-RAF-MEK-ERK was found to be present in about 30% of all human cancer tissues [51].…”

Specificity of Phosphorylation Responses to Mitogen Activated Protein (MAP) Kinase Pathway Inhibitors in Melanoma Cells

“…One docking motif, named "DEF" (aka FRS), binds a pocket near the activation segment of ERK1/2 and typically contains the consensus hydrophobic sequence Phe/Tyr-Xxx-Phe/Tyr located 6-20 residues C-terminal to the phosphorylation site (42,43). A second motif, named "DEJL" (aka Ddomain or DRS), binds a pocket distal to the catalytic cleft in ERK1/2, and often contains the consensus sequence (Arg/Lys)2-3-(Xxx)2-6-ΦA-Xxx-ΦB, where ΦA and ΦB are hydrophobic residues (44). DEJL motifs are more commonly observed, but can be harder to identify because they do not share exact consensus sequences and can vary in their distances from the phosphorylation site (43,45,46).…”

Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo

“…Several years ago, the 1,2,4-triazol-3-one, BI-78D3 was identified as a weak inhibitor of the interaction between the scaffold protein c-Jun N-terminal kinase inhibitory protein 1 (JIP1) and the c-Jun N-terminal kinase 1, JNK1 37 . We found that BI-78D3 impedes the ability of a constitutively active form of the ERK kinase, MKK1 (MKK1G7B) 38 to phosphorylate ERK1 and ERK2 in vitro and also impedes the ability of activated ERK1 and ERK2 to phosphorylate v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1; a construct including the residues 1–138 that is necessary and sufficient for ERK-mediated phosphorylation, was used 39 ) (Fig. 1b).…”