Regulatory Roles of Conserved Phosphorylation Sites in the Activation T-Loop of the MAP Kinase ERK1.

Steven Pelech, Kinexus Bioinformatics Corporation, Vancouver, Canada

The catalytic domains of most eukaryotic protein kinases are highly conserved, and phosphorylation of the activation T-loop, a variable region between the kinase catalytic subdomains VII and VIII, is a common mechanism for stimulation of their phosphotransferase activities. The MAP kinase Extracellular signal-regulated kinase 1 (ERK1) serves as a paradigm for regulation of protein kinases in signaling modules. We investigated the possible roles of three conserved phosphosites in the activation loop of ERK1 flanking the well-documented pTEpY activating site. In vitro kinase assays with myelin basic protein (MBP) using the purified ERK1 phosphosite mutants supported the functional importance of T207 and Y210, but not T198 in regulating ERK1 catalytic activity. Single substitution of the T207 to glutamic acid abolished the activity of ERK1 without affecting the phosphorylation at TEY by MEK1. The Y210 site could be important for proper folding of ERK1 in this regulatory region, since the mutation of this residue caused decreases in protein solubility, and the Y210F mutant was not recognized by MEK1 for phosphorylation in vitro. Our data also indicated that ERK1 autophosphorylated at T207, while the phosphorylation of Y210 was enhanced in presence of MEK1. We hypothesize that following the activation of ERK1, subsequent slower phosphorylation of the flanking sites may result in autoinhibition of the kinase. Hyperphosphorylation within the kinase activation T-loop may serve as a general mechanism for protein kinase down-regulation after initial activation by their upstream kinases.