REGULATION OF EXTRACELLULAR SIGNAL-REGULATED PROTEIN KINASE-1 (ERK-1,PP44 MITOGEN-ACTIVATED PROTEIN-KINASE) BY EPIDERMAL GROWTH-FACTOR ANDNERVE GROWTH-FACTOR IN PC12 CELLS - IMPLICATION OF ERK1 INHIBITORY ACTIVITIES
P. Peraldi et al., REGULATION OF EXTRACELLULAR SIGNAL-REGULATED PROTEIN KINASE-1 (ERK-1,PP44 MITOGEN-ACTIVATED PROTEIN-KINASE) BY EPIDERMAL GROWTH-FACTOR ANDNERVE GROWTH-FACTOR IN PC12 CELLS - IMPLICATION OF ERK1 INHIBITORY ACTIVITIES, Endocrinology, 132(6), 1993, pp. 2578-2585
In PC12 cells, extracellular signal-regulated kinase-1 (ERK1 or pp44/m
itogen-activated protein kinase) is stimulated in response to epiderma
l growth factor (EGF) and nerve growth factor (NGF). This stimulation
is rapid and short-lived after EGF activation. In contrast, NGF promot
es a swift, but persistent, ERK1 stimulation. We took advantage of thi
s difference in activation pattern to study the negative regulation of
ERK1. Using antibodies to the C-terminus of ERK1, we performed in vit
ro reconstitution experiments with immunoprecipitated ERK1 from stimul
ated 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 opp
osite that of ERK1 phosphorylation and activity. Indeed, the highest E
RK1 activation was associated with the lowest ERK1-repressing activity
and vice versa. This ERK1 inhibitory activity was found to be sensiti
ve mainly to sodium orthovanadate and to a lesser extent to zinc aceta
te. Interestingly, okadaic acid decreased ERK1-repressing activity fro
m unstimulated cells when tested with ERK1 from 5-min NGF-treated cell
s, but not with ERK1 from 5-min EGF-treated cells. Hence, ERK1 appears
to be regulated differently after stimulation of cells with EGF compa
red 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 t
reatments of the cells, and that has a profile of regulation similar t
o that of the ERK1 inhibitory activity. This regulatable phosphatase a
ctivity was also observed using in vitro phosphorylated ERK1. Taken to
gether, our data provide evidence that ERK1 is negatively controlled b
y a phosphatase(s) that can undergo differential modulation depending
on the stimuli used.