(Received for publication, January 25, 1996)
From the
Insulin supports the survival and differentiation of many types of fetal neurons. To determine if mitogen-activated protein (MAP) kinases play a role in mediating the neurotrophic actions of insulin, we identified the MAP kinases present in fetal chick forebrain neurons and examined their regulation by insulin. Cell extracts were fractionated on Mono Q columns, and phosphotransferase activity was measured using myelin basic protein as the substrate. In control neurons, four peaks of MAP kinase activity were resolved. Peaks I, II, and IV were identified by immunoblotting as c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 MAP kinase, respectively. Neurons treated with insulin showed a dramatic decrease, 80-90%, in p38 MAP kinase activity without significant changes in the other MAP kinase activities. Insulin decreased the phosphotyrosine content of p38 MAP kinase with maximal effects observed within 5 min. Pretreatment of neurons with sodium orthovanadate blocked the ability of insulin to inhibit the tyrosine phosphorylation and activity of p38 MAP kinase, suggesting that activation of a tyrosine or dual specific phosphatase is necessary for the inhibition of p38 MAP kinase by insulin. Since p38 MAP kinase has been recently implicated in neuronal cell apoptosis, negative regulation of this kinase by insulin may be critical for the neurotrophic actions of insulin.
MAP ()kinase cascades are key signaling systems by
which cells transduce extracellular stimuli into intracellular
responses (for recent reviews see (1) and (2) ). Many
steps of these signaling cascades are conserved in Caenorhabditis
elegans, Drosophila melanogaster, and mammalian cells. In
mammalian cells, the extracellular signal-regulated kinases, ERK1 and
ERK2, are the prototypic members of the MAP kinase family. ERK1 and
ERK2 were originally found to be activated by a number of growth
factors that interact with cell surface tyrosine kinase receptors. The
main features of the ERK cascade involve
p21
-mediated translocation of an upstream serine
kinase, c-Raf, which phosphorylates and activates its downstream
protein kinase, MAP kinase kinase (also referred to as MEK or MKK1).
MAP kinase kinase, a dual specific protein kinase, activates ERK1 or
ERK2, which, in turn, regulates the activity of a variety of cytosolic
enzymes and nuclear transcription factors(3) . Thus, a signal
generated at the cell surface is translated to the nucleus where gene
expression is regulated. Recently, other MAP kinases have been
identified including c-Jun N-terminal kinase (JNK), which is also
referred to as stress-activated protein kinase (4, 5) and p38 MAP kinase, the mammalian homologue of
the yeast HOG1 protein kinase(6, 7) . A characteristic
feature of all the MAP kinases is their activation by phosphorylation
on Tyr and Thr residues within a TXY phosphorylation motif,
where X can be Glu, Pro, or Gly(1, 2) . The
newly discovered MAP kinases, JNK and p38, appear to have equivalent
but distinct upstream kinases that are activated by a wide variety of
extracellular stimuli including growth factors, differentiating
factors, UV irradiation, heat shock, and changes in
osmolarity(4, 5, 6, 7) . In some
cases, a given extracellular signal appears to activate multiple MAP
kinase pathways, whereas other signals appear to activate a single MAP
kinase pathway. In yeast, six distinct MAP kinase pathways have been
identified(8) , and it is likely that many more exist in
mammalian cells.
The MAP kinase cascade involving ERK1 and ERK2 has
been implicated in signal transduction by the insulin receptor. Insulin
activates p21, Raf-1, MEK, and ERK1/ERK2 in a
number of cell types (9) . Activation of this cascade is
mediated by GRB2, a 23-kDa SH2-containing protein, which couples
tyrosine-phosphorylated IRS-1 or SHC (both substrates for the insulin
receptor kinase) to a p21
-specific GDP/GTP
exchange factor(10) . Microinjection of Xenopus oocytes with a neutralizing p21
antibody or
a dominant negative p21
mutant inhibits the
meiotic maturation induced by insulin(11, 12) .
Moreover, overexpression of p21
oncogenes mimics
the effects of insulin on 3T3 cell differentiation, and dominant
negative mutants of p21
block the
differentiation process triggered by insulin(13, 14) .
Taken together, these data suggest that
p21
-mediated activation of ERK1/ERK2 is critical
for certain actions of insulin. However, there is also accumulating
evidence that ERK1/ERK2 activation is cell-specific and may not be
required or sufficient for all of the cellular effects of insulin. In
this study, we examined the regulation of MAP kinases by insulin in
cultured fetal neurons from chick forebrain. Insulin acts as a potent
neurotrophic factor for these neurons (15) and other neurons
from cerebellum, mesencephalon, cerebral cortex, spinal cord, and
hippocampus. Insulin's neurotrophic actions include supporting
the growth of neurons in serum-free medium(16, 17) ,
stimulating neuronal protein synthesis(18) , inducing neurite
outgrowth(16, 17) , and regulating the expression of
certain neurofilament and early immediate
genes(19, 20) . We report here that insulin has no
significant effect on ERK or JNK activities in chick forebrain neurons
but markedly inhibits a high basal level of p38 MAP kinase in these
cells. These results are the first to demonstrate an inhibitory
regulation of a MAP kinase by a growth factor. The inhibition of p38
MAP kinase, a kinase that has recently been linked to neuronal
programmed cell death(21) , may represent the mechanism by
which insulin supports neuronal survival.
To investigate the MAP kinase activities present in fetal neurons, neurons from E8 chick forebrains were cultured for 5 days, and neuronal cell extracts were fractionated by anion exchange chromatography on Mono Q. Phosphotransferase activity was measured in each fraction using MBP as the substrate. Four peaks of MBP kinase activity were resolved (Fig. 1A). The first peak eluted at a NaCl concentration of about 30 mM. This peak contained JNK kinase identified by immunoblotting with anti-JNK antibodies (Fig. 1B) and had the greatest phosphotransferase activity against a GST-c-Jun fusion protein relative to the other fractions (data not shown). Peak II eluted at about 170 mM NaCl and displayed immunoreactivity with an antibody that recognizes the C terminus of both ERK1 and ERK2 (Fig. 1, A and B). The identity of Peak III, which eluted over a wide range of salt concentrations and showed no cross-reactivity with any of the MAP kinase antibodies used, is unknown. Peak IV eluted at about 420 mM NaCl and contained the majority of neuronal MBP kinase activity. Analysis of this peak by MBP-impregnated gel assays revealed a 38-kDa kinase (data not shown), and immunoblot analysis demonstrated positive cross-reactivity with anti-mouse p38 antibodies (Fig. 1, A and B). All four peaks of MBP kinase activity could be absorbed to phenyl-Sepharose, which is a common feature of MAP kinases (data not shown).
Figure 1:
Mono Q column chromatography of control
and insulin-treated neuronal cell extracts. Panel A, neurons
were cultured from E8 chick forebrain. After 5 days in culture, neurons
were incubated in the absence and presence of 50 ng/ml insulin for 15
min at 37 °C. Following solubilization, the cell extracts
(3-5 mg of protein) were fractionated by Mono Q fast protein
liquid chromatography and the fractions assayed for protein kinase
activity using MBP as the substrate. Panel B, column fractions
were concentrated 20-fold using Centricon 10 concentrators (Amicon),
solubilized in 2 Laemmli sample buffer, and analyzed by
SDS-PAGE on 12% acrylamide gels. After electrophoresis, the proteins in
the gel were transferred to PVDF membranes and probed with polyclonal
anti-JNK antibodies (1:100), monoclonal anti-ERK1/ERK2 antibodies
(1:1000), and polyclonal anti-p38 antibodies (1:1000) followed by
secondary antibodies coupled to alkaline phosphatase. Bound alkaline
phosphatase was visualized by the Western-Light (Tropix)
chemiluminescent detection system.
When neurons were treated with insulin (50 ng/ml), a potent neurotrophin for these cells, for 15 min at 37 °C prior to fractionation of cell lysates on Mono Q columns, there was a dramatic and very consistent decrease (80-90%, n = 3) in the activity of Peak IV without major changes in the other MAP kinase activities (Fig. 1A). The inhibition of p38 MAP kinase activity by insulin was specific for this polypeptide since IGF-I at the same concentration had no effect on p38 MAP kinase activity, despite the 10-fold higher number of IGF-I receptors on these neurons. Exposure of the neurons to sorbitol (200 µM), a known activator of p38 MAP kinase(6) , resulted in a 20% increase in Peak IV kinase activity (data not shown). The relatively low stimulation of p38 MAP kinase activity by sorbitol is most likely related to the high basal level of p38 MAP kinase activity in these neurons.
The inhibition of MAP kinase activity by insulin was specific for p38 MAP kinase since the other MAP kinases present were not significantly affected by insulin. This was demonstrated by examining the phosphotransferase activities of the various MAP kinases using the N-terminal half of ATF2 as the substrate (Fig. 2). Insulin inhibited the ATF2 phosphotransferase activity of p38 MAP kinase by 90% with no significant effect on the phosphotransferase activities of the other MAP kinases. ATF2 was observed to be the best substrate for p38 when compared with MBP and the epidermal growth factor receptor peptide (data not shown).
Figure 2:
In vitro phosphorylation of ATF2
by Mono Q column fractions. Column fractions (peaks I, II, III, and IV) from control(-) and insulin-treated
(+) neurons were assayed for kinase activity as described in Fig. 1except that 0.1 mg/ml ATF2 (N-terminal half) was added as
the substrate. Reactions were terminated by the addition of 2
Laemmli sample buffer and analyzed by SDS-PAGE using 10% acrylamide
gels. After electrophoresis, the gels were dried and subjected to
phosphorimaging (GS-100, Bio-Rad).
MAP kinases are activated by phosphorylation of critical tyrosine and threonine residues. To determine if the inhibition of p38 MAP kinase activity by insulin was due to changes in the tyrosine phosphorylation state of the enzyme, control and insulin-treated neuronal cell extracts were immunoprecipitated with anti-phosphotyrosine antibodies (PY20), and the phosphotyrosine-containing proteins in the precipitants were analyzed by Western blotting with anti-p38 antibodies. The results demonstrated that insulin, but not sorbitol or IGF-I, decreased the phosphotyrosine content of p38 (Fig. 3A). The effect of insulin was maximal by 5 min and sustained out to 15 min (Fig. 3, B and C). After 15 min, the phosphotyrosine content of p38 began to approach control levels. Similar results were obtained by immunoprecipitating neuronal cell extracts with p38 antibodies and then subsequently Western blotting with PY20.
Figure 3:
Insulin decreases tyrosine phosphorylation
of p38 MAP kinase. Panel A, neurons were incubated in the
absence or presence of insulin (50 ng/ml), IGF-I (50 ng/ml), and
sorbitol (200 µM) for 15 min at 37 °C. After washing 2
in ice-cold PBS, the cells were solubilized, and cell lysates
were analyzed by immunoprecipitation using PY20 monoclonal anti-Tyr(P)
antibodies. The immunocomplexes were then analyzed by Western blotting
using polyclonal anti-p38 antibodies. Bound horseradish peroxidase was
visualized by the ECL chemiluminescent detection system. IP, immunoprecipitate. Panel B, neurons were incubated in the
absence or presence of insulin (50 ng/ml) at 37 °C for the
indicated times. The cells were solubilized and analyzed as described
in A. Panel C, densitometric scans performed on
experiments (n = 4) described in B.
To determine whether activation of a phosphatase might be involved in the inhibition of p38 MAP kinase by insulin, neurons were treated with sodium orthovanadate (1 mM), an inhibitor of tyrosine and dual specific phosphatases, 10 min prior to the addition of insulin. Sodium orthovanadate had no effect on the basal levels of p38 phosphotyrosine content but totally blocked the ability of insulin to decrease the phosphotyrosine content of p38 MAP kinase (Fig. 4). The decrease in phosphotyrosine content was indicative of decreased kinase activity, suggesting that activation of a tyrosine phosphatase or a dual specific phosphatase is necessary for the inhibition of p38 MAP kinase by insulin. This activated phosphatase may act directly on p38 MAP kinase or at steps upstream of p38 MAP kinase, which are regulated by tyrosine phosphorylation.
Figure 4: Vanadate blocks the insulin-induced decrease in the phosphotyrosine content of p38 MAP kinase. Neurons were pretreated with or without 1 mM sodium orthovanadate 10 min prior to the addition of insulin (50 ng/ml) for 15 min at 37 °C. After washing in ice-cold PBS, the cells were solubilized and subjected to immunoprecipitation with PY20 anti-Tyr(P) antibodies. The immunocomplexes were then analyzed by SDS-PAGE and immunoblotted with anti-p38 antibodies. IP, immunoprecipitate.
Although ERK, JNK, and p38 MAP kinase are structurally related kinases and are stimulated by similar kinase cascades, they are activated by different extracellular stimuli and have different substrate specificities(1, 2) . The p38 MAP kinase was originally described as the mammalian homologue of the Saccharomyces cerevisiae HOG1 kinase involved in osmoregulation in the yeast. In mammalian cells, p38 MAP kinase is activated by changes in osmolarity, bacterial endosomes, and heat shock(6) . Very recently, p38 MAP kinase has been implicated in neuronal programmed cell death(21) . In differentiated PC12 cells, NGF withdrawal results in apoptosis, which is preceded by an increase in p38 MAP kinase activity. Introduction of the dominant negative mutant of MKK3, the upstream regulator of p38 MAP kinase, blocks the apoptosis induced by NGF withdrawal, whereas introduction of the constitutively activated form of MKK3 induced apoptosis in PC12 cells. Thus, stimulation of p38 MAP kinase by NGF withdrawal appears to mediate neuronal programmed cell death. These data, taken together with the results from the present study, suggest that the mechanism by which insulin supports neuronal survival involves inhibition of p38 MAP kinase. This is the first evidence for negative regulation of p38 MAP kinase by a trophic factor and suggests that this MAP kinase may be an important target for other neurotrophic factors.