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

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.