Regulation of extracellular signal-regulated protein kinase-1 (ERK-1; pp44/mitogen-activated protein kinase) by epidermal growth factor and nerve growth factor in PC12 cells: implication of ERK1 inhibitory activities.

In PC12 cells, extracellular signal-regulated kinase-1 (ERK1 or pp44/mitogen-activated protein kinase) is stimulated in response to epidermal growth factor (EGF) and nerve growth factor (NGF). This stimulation is rapid and short-lived after EGF activation. In contrast, NGF promotes a swift, but persistent, ERK1 stimulation. We took advantage of this difference in activation pattern to study the negative regulation of ERK1. Using antibodies to the C-terminus of ERK1, we performed in vitro reconstitution experiments with immunoprecipitated ERK1 from stimulated cells and extracts from PC12 cells incubated with EGF or NGF for various periods of times. Using this approach, we showed that extracts from unstimulated cells reduce ERK1 activity. Upon exposure of cells to NGF or EGF, we found that the inhibitory activity had a pattern opposite that of ERK1 phosphorylation and activity. Indeed, the highest ERK1 activation was associated with the lowest ERK1-repressing activity and vice versa. This ERK1 inhibitory activity was found to be sensitive mainly to sodium orthovanadate and to a lesser extent to zinc acetate. Interestingly, okadaic acid decreased ERK1-repressing activity from unstimulated cells when tested with ERK1 from 5-min NGF-treated cells, but not with ERK1 from 5-min EGF-treated cells. Hence, ERK1 appears to be regulated differently after stimulation of cells with EGF compared to NGF. We show that cell extracts promote ERK1 dephosphorylation. Indeed, we were able to detect a phosphatase activity toward in vivo phosphorylated ERK1 that was regulated differently after NGF and EGF treatments of the cells, and that has a profile of regulation similar to that of the ERK1 inhibitory activity. This regulatable phosphatase activity was also observed using in vitro phosphorylated ERK1. Taken together, our data provide evidence that ERK1 is negatively controlled by a phosphatase(s) that can undergo differential modulation depending on the stimuli used.