Molecular cloning of mouse ERK5/BMK1 splice variants and characterization of ERK5 functional domains

The mitogen-activated protein kinases (MAPKs) play important roles in regul ation of cell growth and survival. Human MAPK 5 (ERK5) or Big MAP kinase 1 (BMK1) is a recently cloned member of the MAPK family. To identify ERK5-rel ated kinases, we searched the GenBank(TM) expressed sequence tag (EST) data base for mouse cDNAs with homology to human ERK5. A full-length mouse cDNA that was highly homologous to the human ERK5 was identified. Further analy sis of ERK5 polymerase chain reaction products generated from mouse embryo cDNA yielded three mouse ERK5 cDNAs (mERK5a, mERK5b, and mERK5c). Sequence analysis showed that these cDNAs are alternative splice products of the mou se ERK5 gene. Interestingly, expressed mERK5b and mERK5c act as dominant ne gative inhibitors based on inhibition of mERK5a kinase activity and mERK5a- mediated MEF2C transactivation. However, the physiological significance of mERK5b and mERK5c is not fully understood. Further investigation using thes e mouse ERK5 splice variants and other constructed mutants identified funct ional roles of several regions of mERK5, which-appear to be important for p rotein-protein interaction and intracellular localization. Specifically, we found that the long C-terminal tail, which contains a putative nuclear loc alization signal, is not required for activation and kinase activity but is responsible for the activation of nuclear transcription factor MEF2C due t o nuclear targeting. In addition, the N-terminal domain spanning amino acid s (aa) 1-77 is important for cytoplasmic targeting; the domain from aa 78 t o 139 is required for association with the upstream kinase MEK5; and the do main from aa 140-406 is necessary for oligomerization. Taken together, thes e observations indicate that ERK5 is regulated by distinct mechanisms deter mined by its unique structure and presumably the presence of multiple splic e variants.