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ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK3 (MAPK6) are atypical MAPKs. One major difference between these proteins and the classical MAPKs is substitution of the conserved T-X-Y motif within the activation loop by a single phospho-acceptor site within an S-E-G motif. In the present study we report that Ser186 of the S-E-G motif in ERK4 is phosphorylated in vivo. Kinase-dead ERK4 is also phosphorylated on Ser186, indicating that an ERK4 kinase, rather than autophosphorylation, is responsible. Co-expression of MK5 [MAPK-activated protein kinase 5; also known as PRAK (p38-regulated/activated kinase)], a physiological target of ERK4, increases phosphorylation of Ser186. This is not dependent on MK5 activity, but does require interaction between ERK4 and MK5 suggesting that MK5 binding either prevents ERK4 dephosphorylation or facilitates ERK4 kinase activity. ERK4 mutants in which Ser186 is replaced with either an alanine residue or a phospho-mimetic residue (glutamate) are unable to activate MK5 and Ser186 is also required for cytoplasmic anchoring of MK5. Both defects seem to reflect an impaired ability of the ERK4 mutants to interact with MK5. We find that there are at least two endogenous pools of wild-type ERK4. One form exhibits reduced mobility when analysed using SDS/PAGE. This is due to MK5-dependent phosphorylation and only this retarded ERK4 species is both phosphorylated on Ser186 and co-immunoprecipitates with wild-type MK5. We conclude that binding between ERK4 and MK5 facilitates phosphorylation of Ser186 and stabilization of the ERK4–MK5 complex. This results in phosphorylation and activation of MK5, which in turn phosphorylates ERK4 on sites other than Ser186 resulting in the observed mobility shift. [ABSTRACT FROM AUTHOR]