From the
The receptors for insulin-like growth factor 1 (IGF1) and
insulin are related heterotetrameric proteins which, like the epidermal
growth factor (EGF) receptor, possess intrinsic ligand-stimulated
tyrosine protein kinase activity. In Rat 1 fibroblasts, stimulation of
mitogen-activated protein (MAP) kinase via the IGF1 receptor and the
G
Activation of the ubiquitous mitogen-activated protein
(MAP)
Recent work has
shown that some G protein-coupled receptors (e.g. the
lysophosphatidic acid (LPA) receptor) which interact with Bordetella pertussis toxin (PT)-sensitive G proteins can also
promote p21
Insulin and insulin-like growth factor 1 (IGF1), in contrast to EGF,
behave as relatively weak mitogens. Their receptors are closely related
proteins which share an
Transient
transfection of Rat 1 cells was performed using LipofectAMINE (Life
Technologies, Inc.). Briefly, confluent monolayers in 6-well tissue
culture plates were incubated at 37 °C with a transfection mixture
composed of 1 ml of serum-free DMEM containing 1.2 µg of DNA/well
and 7 µl of LipofectAMINE. After 2 h, the mixture was aspirated and
replaced with 2 ml of DMEM containing 10% fetal bovine serum. Assays
were performed 48 h after transfection. Empty pRK5 vector was added to
transfections as needed to keep the total mass of DNA added per well
constant within an experiment. Coexpression of the
Several authors have reported that the
PT-sensitive activation of MAP kinase by G protein-coupled receptors is
also sensitive to inhibitors of tyrosine protein kinases (6, 7) and is independent of
PKC(3, 4, 5, 6, 7, 8) .
As shown in Fig. 2A, preincubation of cells with the
tyrosine kinase inhibitor genistein resulted in significant inhibition
of both LPA- and IGF1-mediated MAP kinase phosphorylation at
concentrations (100 µM) which did not impair EGF- or
direct PKC-mediated effects. At higher concentrations (300
µM), EGF-, LPA-, and IGF1-mediated, but not PKC-mediated,
MAP kinase phosphorylation was inhibited (data not shown). MAP kinase
activation resulting from expression of the constitutively activated
mutant p21
We have characterized the activation of MAP kinase by
tyrosine kinase growth factor receptors, G
Insulin and
IGF1 receptor-mediated p21
Interaction between G
Previous work has suggested that in certain cell
types, PT-sensitive G proteins may play a role in several aspects of
insulin receptor
signaling(32, 33, 34, 35) . The
mechanism whereby the insulin or IGF1 receptor might promote the
generation of free G
The data in this report suggest that at
endogenous levels of receptor expression, the tyrosine kinase IGF1
receptor and G
We thank S. T. Exum for excellent technical assistance
and M. Holben and D. Addison for secretarial assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-coupled receptor for lysophosphatidic acid (LPA), but not
via the EGF receptor, is sensitive both to pertussis toxin treatment
and to cellular expression of a specific G
subunit-binding peptide. The IGF1, LPA, and EGF receptor-mediated
signals are all sensitive to inhibitors of tyrosine protein kinases,
require p21
activation, and are independent of
protein kinase C. These data suggest that some tyrosine kinase growth
factor receptors (e.g. IGF1 receptor) and classical G
protein-coupled receptors (e.g. LPA receptor) employ a similar
mechanism for mitogenic signaling that involves both tyrosine
phosphorylation and G
subunits derived from
pertussis toxin-sensitive G proteins.
(
)kinase pathway is thought to proceed
through a defined sequence of protein phosphorylations and
protein-protein interactions. The best understood example is that
triggered by the receptor for epidermal growth factor (EGF). Ligand
binding promotes EGF receptor dimerization and tyrosine
autophosphorylation. Assembly of a multiple protein complex at the cell
membrane directed by Src homology (SH) 2 and SH3 domain interactions
results in p21
GTP-exchange and activation of
the Raf-1 kinase. In the ensuing phosphorylation cascade, activation of
MAP kinases follows their phosphorylation by the mixed function
threonine/tyrosine kinase, MEK (MAP kinase/extracellular
signal-regulated kinase)(1, 2) .
-dependent activation of MAP
kinase(3, 4, 5, 6, 7, 8) .
This pathway is sensitive to inhibitors of tyrosine protein kinases (6, 7, 9) and requires G protein
G
subunits(3, 10, 11) .
heterotetrameric structure and, like the EGF receptor, possess
ligand-stimulated tyrosine kinase activity(12) . Moreover,
receptor-catalyzed tyrosine phosphorylation of exogenous proteins, such
as insulin receptor substrate 1 (IRS-1) (13, 14) or
Shc(15) , is one of the earliest steps in mitogenesis triggered
by these receptors. Once phosphorylated, these proteins are felt to
function as platforms for assembly of the mitogenic signaling protein
complex, which then proceeds following the EGF receptor paradigm. Here
we present the surprising observation that, in contrast to the
classical EGF receptor paradigm, stimulation of the MAP kinase pathway
by the tyrosine kinase IGF1 receptor also requires the participation of
G
subunits derived from PT-sensitive G proteins.
Like the G protein-coupled receptor-mediated pathway, the IGF1 signal
can be blocked by either PT treatment or by an inhibitor of
G
subunit-mediated signaling. Thus, mitogenic
signals originating from this class of tyrosine kinase growth factor
receptors converge with the G protein-coupled receptor signaling
pathway at a point upstream of p21
activation.
DNA Constructs
The cDNA encoding
hemagglutinin-tagged p44 (p44
) in the
pcDNA1 eukaryotic expression plasmid was provided by J.
Pouysségur. The dominant negative mutant
p21
in the pRS
eukaryotic
expression plasmid was provided by D. Altschuler and M. Ostrowski.
Cell Culture and Transfection
Rat 1 cells were
maintained in Dulbecco's modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin
at 37 °C in a humidified, 5% CO atmosphere. Stably
transfected
ARK1-CT peptide-expressing Rat 1 mutant 277 cells (10) were maintained under identical conditions in the presence
of 1000 µg/ml Geneticin (Life Technologies, Inc.).
ARK1-CT peptide
was confirmed by protein immunoblotting of whole cell detergent lysates
using rabbit anti-
ARK1 carboxyl terminus serum as
described(16) . Endogenous IGF1 receptor expression was
quantified by equilibrium binding of
I-IGF1 (DuPont NEN)
as described(17) .
Measurement of MAP Kinase Phosphorylation State and
Kinase Activity
Agonist-stimulated phosphorylation of endogenous
p42 (erk2) was determined by electrophoretic
mobility shift as described(10) . Activation of p44
(erk1) was measured using a modification of the
procedure of Meloche et al.(18) . Transfected Rat 1
cells were preincubated overnight in serum-free medium and stimulated
for 5 min (IGF1, EGF, and PMA) or 10 min (LPA). Monolayers were washed
with ice-cold calcium- and magnesium-free phosphate-buffered saline,
lysed in 200 µl of ice-cold lysis buffer (50 mM Tris-Cl,
pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% v/v Nonidet P-40,
0.5% w/v sodium deoxycholate, 0.1% SDS, 10 mM NaF, 10 mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonyl
fluoride), and clarified by centrifugation. Coexpressed p44
was immunoprecipitated from the supernatant using 6.5 µg of
12CA5 antibody and 30 µl of a 50% slurry of protein A-agarose.
Washed immune complexes were resuspended in 40 µl of kinase buffer
(20 mM HEPES, pH 7.4, 10 mM MgCl
, 1
mM dithiothreitol) containing 250 µg/ml myelin basic
protein (MBP), 20 µM ATP, 2.5 µCi of
[
-
P]ATP and incubated for 20 min at 30
°C. The reaction was terminated by the addition of 40 µl of 2
Laemmli sample buffer, and
P-labeled MBP was
resolved by SDS-PAGE. Phosphorylation was quantitated using a Molecular
Dynamics PhosphorImager.
Effects of Pertussis Toxin Treatment, Tyrosine Kinase
Inhibitors, and PKC Depletion on IGF1 Receptor-mediated MAP Kinase
Activation
Fig. 1depicts the effects of PT treatment on
the time course of ligand-stimulated phosphorylation of MAP kinase
mediated by endogenous receptors in wild-type Rat 1 cells.
Lysophosphatidic acid (LPA), the simplest naturally occurring
phospholipid, provoked a transient increase in MAP kinase
phosphorylation, which was abolished by PT pretreatment (Fig. 1A). MAP kinase phosphorylation in response to
insulin or IGF1 exposure followed a similar time course and was also
PT-sensitive (Fig. 1, B and C). In contrast,
MAP kinase phosphorylation stimulated by platelet-derived growth
factor, acidic fibroblast growth factor, or EGF (Fig. 1, D-F), each of which is mediated by membrane receptors
with intrinsic tyrosine kinase activity, exhibited a more robust and
sustained activation which was not significantly affected by PT
treatment. A similar pattern of PT sensitivity was observed in
C3H10T1/2 murine fibroblasts. Thus, the responses mediated by tyrosine
kinase growth factor receptors apparently segregated into two groups:
one PT-sensitive, resembling the Gi-coupled LPA receptor, and
one PT-insensitive.
Figure 1:
Effect of PT treatment on MAP kinase
activation via endogenously expressed receptors in Rat 1 cells. Rat 1
cells, cultured as described (15), were incubated overnight in
serum-free medium in the presence or absence of PT (100 ng/ml) prior to
determination of agonist-stimulated phosphorylation of endogenous
p42 as described. Upper panel, representative
autoradiograph demonstrating the effects of PT treatment on the
p42
phosphorylation state following a 5-min stimulation
with LPA (10 µM), IGF1 (100 ng/ml), or EGF (10 ng/ml). Lower panel, the time course of agonist-induced
phosphorylation of MAP kinase in control (
) and PT-treated
(
) cells was determined following stimulation with LPA (A), insulin (100 nM) (B), IGF1 (C), platelet-derived growth factor (10 ng/ml) (D),
fibroblast growth factor (10 ng/ml) (E), or EGF (F).
Data are presented as the percent of total p42
present
in the phosphorylated state. Each point represents the mean ±
S.E. for three to six separate experiments.
Insulin, IGF1, and insulin-like growth factor 2
(IGF2) have distinct cell surface receptors, each of which can bind
insulin, IGF1, and IGF2 with varying affinity. The receptors can be
discriminated based upon the rank order of potency displayed by each
agonist. The receptor for IGF2 consists of a single membrane-spanning
polypeptide that lacks intrinsic tyrosine kinase activity(19) .
While not considered a mediator of mitogenic signals, the IGF2 receptor
can reportedly interact with PT-sensitive G proteins(20) .
Dose-response curves for MAP kinase phosphorylation in Rat 1 cells
revealed a rank order of potency IGF1 > IGF2 insulin (data not
shown), indicating that the effects of all three agonists were mediated
predominantly by IGF1 receptors, as has been previously
reported(21) .
(22) was
unaffected by genistein concentrations up to 300 µM,
(
)indicating that the tyrosine kinase
inhibitor-sensitive step lies upstream of p21
activation. In contrast to the effects of genistein, the
potent tyrosine kinase and PKC inhibitor staurosporine nonspecifically
inhibited MAP kinase activation by each agent.
Figure 2:
Effects of protein kinase inhibitors and
PKC depletion on MAP kinase phosphorylation in Rat 1 cells. A,
serum-starved Rat 1 cells were preincubated for 15 min with genistein
(100 µM) or staurosporine (1 µM) prior to
stimulation for 5 min with LPA, IGF1, EGF, or the PKC activating
phorbol ester, phorbol 12-myristate 13-acetate (PMA) (1
µM). Basal (NS) and agonist-stimulated
phosphorylation of p42 was determined as described. B, Rat 1 cells were preincubated overnight in serum-free
medium in the presence or absence of PMA (0.1 µM) to
down-regulate cellular PKC expression (14), preincubated for 15 min in
the absence of PMA, and stimulated for 5 min with LPA, IGF1, EGF, or
PMA. Phosphorylation of p42
was then determined as
described. Each column represents mean ± S.E. values for percent
of total p42
present in the phosphorylated state in
three separate experiments performed in duplicate. * signifies value
less than control, p < 0.05.
Fig. 2B depicts the effects of cellular PKC depletion on agonist-induced
MAP kinase phosphorylation. Rechallenge with phorbol ester following
PKC depletion provoked no increase in MAP kinase phosphorylation. In
contrast, the effects of LPA, IGF1, and EGF were not significantly
inhibited. These data suggest that stimulation of MAP kinase
phosphorylation by the G-coupled LPA receptor and the
tyrosine kinase IGF1 receptor are qualitatively similar; each is
sensitive to inhibitors of tyrosine kinases and is independent of PKC
activation.
Effect of the G
Recently, G protein
GSubunit-sequestrant
ARK1-CT Peptide on IGF1
Receptor-mediated MAP Kinase Activation
subunits have been implicated in the direct
p21
-dependent activation of MAP kinase mediated
by PT-sensitive G protein-coupled receptors including the LPA receptor.
Coexpression of G
subunits in COS-7 cells leads
directly to MAP kinase activation(3, 11) . We have
previously shown that cellular expression of a specific
G
subunit binding peptide derived from the
carboxyl terminus of the
-adrenergic receptor kinase 1
(
ARK1-CT) (23, 24) specifically antagonizes
G
subunit-mediated MAP kinase activation (10, 25) in stably and transiently transfected cell
lines. To determine whether the PT-sensitive phosphorylation of MAP
kinase stimulated by the IGF1 receptor was mediated by G
subunits, we studied the effects of IGF1 in a Rat 1 cell line
which stably overexpressed the
ARK1-CT peptide (277
cells)(10) . As shown in Fig. 3, compared with the
parental Rat 1 cells, LPA-stimulated MAP kinase phosphorylation was
attenuated by about 50% (Fig. 3A). IGF1-stimulated
phosphorylation was undetectable in the 277 cells (Fig. 3B), while the EGF signal was unaffected (Fig. 3C). Thus, sensitivity to inhibition by PT and a
G
subunit-binding peptide are properties shared by
the LPA- and IGF1-mediated pathways.
Figure 3:
Time course of agonist-stimulated MAP
kinase phosphorylation Rat 1 cells which stably overexpress the
G subunit-binding
ARK1-CT peptide.
ARK1-CT peptide-expressing Rat 1 mutant 277 cells (15) were
incubated overnight in serum-free medium in the presence (
) or
absence (
) of PT prior to determination of the time course of
agonist-induced phosphorylation of endogenous p42
following stimulation with LPA (A), IGF1 (B),
or EGF (C). The time course observed in the parental Rat 1
cells (
) is shown for reference. Data are presented as the percent
of total p42
present in the phosphorylated state. Each
point represents the mean ± S.E. for three separate experiments.
Endogenous IGF1 receptor density was 8.5-10.0 fmol/mg of whole
cell protein in both the parental Rat 11 and 277 cell
lines.
To confirm that the inhibition
of MAP kinase phosphorylation by the G subunit
antagonist reflected impaired kinase activation, MAP kinase activity
was determined in Rat 1 cells transiently cotransfected with a peptide
minigene encoding the
ARK1-CT peptide and hemagglutinin-tagged
p44
(18). Kinase activity of immunoprecipitated
p44
following stimulation was determined to assess
MAP kinase activation in the transfected cell pool. As shown in Fig. 4A, results of the p44
kinase
assay resembled the findings in 277 cells. LPA and IGF1
receptor-mediated MAP kinase activation was significantly attenuated
both by PT treatment and by expression of the
ARK1-CT peptide; the
EGF receptor signal, as well as that produced by direct activation of
PKC, was not affected.
Figure 4:
Effects
of PT treatment, ARK1-CT peptide, and dominant negative
p21
expression on MAP kinase activity in transiently
transfected Rat 1 cells. A, Rat 1 cells were transiently
transfected with hemagglutinin-tagged p44
(24) with
or without a peptide minigene encoding the
ARK1-CT peptide (15,
23). The effects of PT treatment and
ARK1-CT peptide expression on
basal (NS) and agonist-stimulated MAP kinase activity,
assessed as phosphorylation of myelin basic protein (MBP) by
immunoprecipitated p44
, was determined following
exposure to LPA, IGF1, EGF, or PMA. B, Rat 1 cells were
transiently transfected with hemagglutinin-tagged p44
with or without a plasmid encoding the dominant negative mutant
p21
(41). Basal (NS) and agonist-stimulated MAP
kinase activity was determined following exposure to LPA, IGF1, EGF, or
PMA. MBP phosphorylation is presented in arbitrary units such that 1
unit equals the amount of [
P]phosphate
incorporated in unstimulated control cells. Data shown represent mean
± S.E. values for three separate experiments performed in
duplicate. * signifies value less than control, p <
0.05.
The pathways of both G-coupled
receptor- and EGF receptor-mediated mitogenesis can also be
discriminated from the PKC-dependent pathway based upon their
dependence upon activation of p21
GTP-exchange.
As shown in Fig. 4B, transient coexpression of the
dominant negative mutant p21
in
Rat 1 cells resulted in inhibition of LPA, IGF1, and EGF
receptor-stimulated p44
activity, with no significant
effect upon phorbol ester-induced activation.
-coupled
receptors, and phorbol ester in wild-type and mutant Rat 1 fibroblasts.
The data suggest that two tyrosine kinase growth factor receptors,
those for EGF and IGF1, employ distinct mechanisms of MAP kinase
activation. Unlike the well described EGF receptor pathway, activation
of MAP kinase by the IGF1 receptor proceeds via a G
subunit-dependent activation of p21
. This
pathway has previously been implicated only in MAP kinase activation
mediated by classical G
-coupled receptors.
activation follows
the formation of a protein complex involving the growth factor
receptor-binding protein, GRB2, the guanine nucleotide exchange factor,
Sos1, and tyrosine-phosphorylated Shc or
IRS-1(13, 14, 15) . The molecular mechanism of
G
subunit-dependent p21
activation is unknown. Recently, however, the
G
-coupled receptors for thyrotropin-releasing factor (9) and endothelin 1 (26) have been shown to mediate
tyrosine phosphorylation of Shc and Shc-GRB2 complex formation and the
-thrombin receptor to activate the
p60
tyrosine kinase(27) .
These observations, along with the data presented here, suggest that G
protein-coupled receptors and some tyrosine kinase growth factor
receptors may share a common pathway of mitogenic signal transduction.
subunits and the carboxyl
terminus of
ARK is responsible for agonist-induced translocation
of the kinase from the cytosol to the plasma membrane, where it
initiates the process of homologous
desensitization(23, 24) . It is tempting to speculate
that a similar G
subunit-mediated protein
translocation or localization event, involving some component of the
mitogenic signaling complex, facilitates transmission of receptor
signals to p21
and MAP kinase. The
G
subunit-binding carboxyl terminus of
ARK
contains a region of protein sequence homology, termed a pleckstrin
homology (PH) domain (28, 29), which is also shared by several proteins
which participate in the regulation of p21
. In vitro binding of G
subunits to PH
domain-containing peptides derived from several of these proteins,
including Ras-GRF, Ras-GAP, IRS-1(30) , and the Bruton tyrosine
kinase(31) , has been reported. Thus, noncovalent interactions
between G
subunits and the PH domains of one or
more proteins involved in the regulation of p21
may provide the structural basis for G
subunit-mediated activation of the MAP kinase pathway. In this
regard, it is noteworthy that in transfected Rat 1 cells which stably
overexpress human insulin receptors at high density, we have found that
insulin treatment provokes a more robust and sustained phosphorylation
of MAP kinase which, like the EGF receptor signal, is insensitive to PT
treatment or
ARK1-CT peptide expression (data not shown). This may
reflect an ability of insulin receptors at high density to utilize
another, G
subunit-independent pathway.
Alternatively, it may indicate that G
subunits are
required for efficient signal transduction at low endogenous levels of
receptor expression, but that high receptor density surmounts the
requirement for a G
subunit-directed protein
localization event.
subunits is unclear, although
indirect evidence suggests that a direct protein-protein interaction
may occur between receptor and G protein (36-39). Recently,
peptides derived from the insulin receptor
subunit have been
shown to directly activate G
in phospholipid
vesicles(40) .
-coupled LPA receptor each require both
tyrosine phosphorylation and the release of G
subunits for MAP kinase activation. Understanding how mitogenic
signals generated by these divergent classes of membrane receptor
interact may be of central importance to the development of strategies
to modulate cellular proliferative responses to injury such as those
mediated by platelet-derived mitogens that bind to G protein-coupled
receptors or the accelerated microvascular and macrovascular disease
associated with hyperinsulinemia in patients with diabetes mellitus.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.