MKK5 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) in vitro. Activation was accompanied by the phosphorylation of Thr219 and Tyr221, the former residue being phosphorylated preferentially. ERK5cat phosphorylated at Thr219, but not Tyr221, possessed 10% of the activity of the doubly phosphorylated protein towards myelin basic protein, whereas ERK5cat phosphorylated at Tyr221 alone was much less active. Activated ERK5 phosphorylated itself at a number of residues, including Thr28, Ser421, Ser433, Ser496, Ser731 and Thr733. ERK5 phosphorylated at Thr219, but not Tyr221, phosphorylated itself at a similar rate to ERK5 phosphorylated at both Thr219 and Tyr221. Activated ERK5 also phosphorylated mitogen-activated protein kinase kinase 5 (MKK5) extensively at Ser129, Ser137, Ser142 and Ser149, which are located within the region in MKK5 that is thought to interact with ERK5.

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