Big mitogen-activated protein kinase 1/extracellular-regulated kinase 5 (BMK1/ERK5) is regulated sequentially by a series of upstream MAP kinase kinases (MEKs) in a signaling cascade. MEKs activate their downstream MAPK by phosphorylation of threonine and tyrosine in the T- X-Y motif. MEK5 is the upstream BMK1 kinase and exists as naturally occurring splice variants, MEK5alpha and MEK5beta. The full-length MEK5 (MEK5alpha) is 89 amino acids longer than MEK5beta at the N terminus, but the precise functional difference between the two splice variants is not known. Dual phosphorylation site mutation of MEK5alpha (Ser-311 --> Asp and Thr- 315 --> Asp; MEK5alpha(S311D/T315D)) activated BMK1, but the corresponding dual phosphorylation sites mutant of MEK5beta could not induce BMK1 kinase activation or nuclear translocation. Furthermore, MEK5beta inhibited epidermal growth factor-induced BMK1 activation and MEK5alpha(S311D/T315D)-induced MEF2 transcriptional activity. Both MEK5alpha and MEK5beta individually co-immunoprecipitated with BMK1, but the presence of MEK5beta prevented association of MEK5alpha with BMK1 suggesting a mechanistic basis for the dominant-negative behavior of MEK5beta on BMK1 activation. The ratio of MEK5alpha to MEK5beta expression was higher in cancer cell lines, and overexpression of MEK5beta-inhibited serum-induced DNA synthesis. These data suggest that alternative splicing of MEK5alpha and MEK5beta may play a critical role in BMK1 activation and subsequent cell proliferation.