Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway

Antibodies that recognise the active phosphorylated forms of mitogen-activated protein kinase (MAPK) kinase 5 (MKK5) and extracellular signal-regulated kinase 5 (ERK5) in untransfected cells have been exploited to show that the epidermal growth factor (EGF)-induced activation of MKK5 and ERK5 occurs subsequent to the activation of ERK1 and ERK2 in HeLa cells. The drugs U0126 and PD184352, which prevent the activation of MKK1 (and hence the activation of ERK1/ERK2), also prevent the activation of MKK5, although higher concentrations are required. Our studies define physiological targets of the MKK5/ERK5 pathway as proteins whose phosphorylation is largely prevented by 10 μM PD184352, but unaffected by 2 μM PD184352. Surprisingly, 2 μM PD184352 prolongs the activation of MKK5 and ERK5 induced by EGF or H₂O₂, indicating negative control of the MKK5/ERK5 pathway by the classical MAPK cascade. Our results also indicate that ERK5 is not a significant activator of MAPK-activated protein kinase-1/RSK in HeLa cells.