Functional Significance of Extracellular Signal Regulated Kinase (ERK2) Phosphorylation States: Implications for DNA Binding

The protein kinase extracellular signal-regulated kinase 2 (ERK2) has been well understood structurally for nearly twenty years. New insight is emerging about its structure and function. A novel autophosphorylation has recently been found to occur on a critical active site residue, threonine 188. Autophosphorylation of this residue has been suggested to occur as a result of the confluence of receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) signaling pathways. The possibility of autophosphorylation on threonine 188 seemed to be inconsistent with what was known about the structure and function of protein kinases generally and ERK2 in particular; T188 and the comparable residue in other protein kinases is required for catalytic activity. I found ERK2 phosphorylated on T188 in vitro in partially purified preparations of the recombinant protein purified following expression in bacteria. This suggests that phosphorylation of ERK2 on T188 can occur without the input of upstream RTK or GPCR signaling. Through mutagenesis experiments, I found mutation of T188 sharply reduced activity toward substrates in vitro. Protein fractions containing pT188 and purified ERK2 T188D and T188E mutants, that may act as phosphomimetics, appear to have an increased affinity for DNA binding. I examined critical residues in the activation loop important for phosphorylation and activation of ERK2 and found that perturbation of these residues influences DNA binding specificity. Although the MAPK pathway and role of ERK2 is well understood, our data suggest that previously unrecognized, higher-order signaling mechanisms that arise from additional phosphorylation events may be involved in less well characterized properties of ERK2. Second, our finding that T188 autophosphorylation can occur in recombinant ERK2 independently from upstream GPCR signaling illustrates the need for a reinvestigation of the regulation of ERK2 autophosphorylation. Finally, direct and specific DNA binding may be driven by differences in phosphorylation. The result of this work serves to redefine a most important signaling molecule in terms of its structural modifications and relationship to overall function.