A structure/function analysis of macromolecular recognition by the protein kinase ERK2

Mitogen-activate protein kinases (MAPKs) phosphorylate protein substrates in the presence of magnesium and adenosine triphosphate in response to extracellular environmental signals to carry out signal-dependent intracellular responses. Extracellular signal-regulated protein kinase 2 (ERK2), a member of the MAPK family, mediates cellular growth, differentiation, and proliferation in response to growth factors. Understanding the mechanism by which MAPKs specifically recognize their protein substrates to carry out phosphoryl-transfer on specific residues within these macromolecules is critical for understanding the mechanism of signal transduction fidelity. Phage display was carried out against the active form of ERK2 to find novel ERK2-binding peptides. One peptide, KKKIRCIRGWTKDIRTLADSCQY, inhibited ERK2 phosphorylation of the protein substrate Ets∆138, exhibiting competitive and mixed inhibition towards Ets∆138 (Ki = 20.7 ± 5.5 µM) and MgATP2-, respectively. Steady-state kinetics combined with a novel fluorescence anisotropy binding assay were used to quantitatively elucidate the roles of several proposed ERK2 exosites in forming a macromolecular docking complex with Ets∆138 required for efficient phosphorylation. An ERK2–Ets∆138 docking complex (Kd of 6.6 ± 1.2 µM) was shown to form independent of the substrate phospho-acceptor. Docking motif peptides proposed to bind ERK2 exosites could dissociate the ERK2–Ets∆138 docking complex, however, dissociation did not occur using a peptide containing an ERK2 phospho-acceptor indicating the lack of active site interactions in the docking complex. Mutation of ERK2 residues Lys-229 and His-230 to p38 MAPKα-like residues, an enzyme that does not efficiently phosphorylate Ets∆138, led to a 20-fold decrease in the specificity constant (kcat/Km) of Ets∆138 phosphorylation largely due to its inability to bind Ets∆138. This structure/function analysis offers a quantitative approach towards understanding the molecular determinants of protein substrate recognition by a protein kinase prior to phosphorylation.