An analysis of the phosphorylation and activation of extracellular-signal-regulated protein kinase 5 (ERK5) by mitogen-activated protein kinase kinase 5 (MKK5) in vitro

摘要

pMKK5 expressed as a glutathione S-transferase fusion protein in human embryonic kidney 293 cells activated full-length extracellular-signal-regulated protein kinase (ERK)5 (ERK5wt) as well as the isolated catalytic domain (ERK5cat) iin vitro/i. Activation was accompanied by the phosphorylation of Thrsup219/sup and Tyrsup221/sup, the former residue being phosphorylated preferentially. ERK5cat phosphorylated at Thrsup219/sup, but not Tyrsup221/sup, possessed 10% of the activity of the doubly phosphorylated protein towards myelin basic protein, whereas ERK5cat phosphorylated at Tyrsup221/sup alone was much less active. Activated ERK5 phosphorylated itself at a number of residues, including Thrsup28/sup, Sersup421/sup, Sersup433/sup, Sersup496/sup, Sersup731/sup and Thrsup733/sup. ERK5 phosphorylated at Thrsup219/sup, but not Tyrsup221/sup, phosphorylated itself at a similar rate to ERK5 phosphorylated at both Thrsup219/sup and Tyrsup221/sup. Activated ERK5 also phosphorylated mitogen-activated protein kinase kinase 5 (MKK5) extensively at Sersup129/sup, Sersup137/sup, Sersup142/sup and Sersup149/sup, which are located within the region in MKK5 that is thought to interact with ERK5./p