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(Received for publication, February 21,
1996; and in revised form, April 5, 1996) From the
The
The cellular signaling pathways leading to receptor-tyrosine
kinase- (RTK) ( The signaling pathways
utilized by G Recent studies have suggested that phosphatidylinositol
3-kinase (PI-3K) activity may be involved in both RTK- and
GPCR-mediated mitogenic
signaling(22, 23, 24, 25, 26, 27, 28, 29) .
However, the role of PI-3K in mitogenic signaling pathways has not been
clearly elucidated. In this study, we assess the role of PI-3K in the
G
The
Figure 1:
Effect of PI-3K inhibitors on
G
A recent
study (36) reported that expression of a dominant negative
mutant of p85
Figure 2:
Effect of
In PC12 cells,
GTP-dependent association of Ras with the catalytic subunit of PI-3K
has been described(23) , suggesting that activation of the
p85/p110 PI-3K occurs subsequent to Ras activation. In contrast, a
constitutively active mutant of PI-3K stimulates Ras-dependent Xenopus oocyte maturation and fos transcription (37) suggesting PI-3K activation precedes Ras activation in
this system. To determine whether PI-3K activation in the
G
Figure 3:
Effect of PI-3K inhibitors on LPA- and
EGF-mediated Ras activation. COS-7 cells were preincubated for 15 min
with wortmannin (1.0 µM), LY294002 (20 µM),
or vehicle. Cells were then treated for 2 min with vehicle, LPA (10
µM), or EGF (10 ng/ml), and Ras activation was determined.
Data are shown as GTP bound to Ras as a percent of the total guanyl
nucleotides bound to Ras. Values represent the mean ± S.E. from
three separate experiments.
Further evidence that PI-3K activation is an
early event in the G
Figure 4:
Effect of PI-3K inhibitors on MAP kinase
(HA-ERK1) activation stimulated by intermediates of the
G
In CHO-K1 cells,
coexpression of G A role for PI-3K in mitogenic
signaling has been suggested by previous studies showing that PI-3K can
associate with activated RTKs and Src family
kinases(38, 39, 40) . PI-3K association with
the Grb2-Sos complex has been demonstrated following monocyte
colony-stimulating factor stimulation of human peripheral blood
monocytes (41) and increased PI-3K/Shc association in cells
transformed by BCR/abl oncoprotein has been reported(42) .
Thus, PI-3K is capable of interacting with many mitogenic signaling
intermediates. Several studies have suggested a role for PI-3K in
GPCR-mediated signaling. Activation of neutrophils by
formylated-Met-Leu-Phe involves pertussis toxin-sensitive increases in
PI-3K activity and PIP We have previously observed that G It has recently been reported that a product of PI-3K
activity, phosphatidylinositol 3,4,5-trisphosphate (PIP The ability of
Volume 271,
Number 21,
Issue of May 24, 1996 pp. 12133-12136
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-mediated
Mitogen-activated Protein Kinase Signaling Pathway (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-subunit of G
mediates
mitogen-activated protein (MAP) kinase activation through a signaling
pathway involving Shc tyrosine phosphorylation, subsequent formation of
a multiprotein complex including Shc, Grb2, and Sos, and sequential
activation of Ras, Raf, and MEK. The mechanism by which G
mediates tyrosine phosphorylation of Shc, however, is unclear. This
study assesses the role of phosphatidylinositol 3-kinase (PI-3K) in
G-mediated MAP kinase activation. We show that
G-coupled receptor- and G-stimulated MAP kinase
activation is attenuated by the PI-3K inhibitors wortmannin and
LY294002 or by overexpression of a dominant negative mutant of the p85
subunit of PI-3K. Wortmannin and LY294002 also inhibit
G-coupled receptor-stimulated Ras activation. The PI-3K
inhibitors do not affect MAP kinase activation stimulated by
overexpression of Sos, a constitutively active mutant of Ras, or a
constitutively active mutant of MEK. These results demonstrate that
PI-3K activity is required in the G-mediated MAP kinase
signaling pathway at a point upstream of Sos and Ras activation.
)and G protein-coupled receptor- (GPCR)
stimulated mitogen-activated protein (MAP) kinase activation have
recently been the subject of intense investigation (1, 2, 3, 4, 5, 6) .
The signaling pathway of RTK-mediated MAP kinase activation is the most
clearly understood. Epidermal growth factor (EGF) stimulation, for
example, produces activation and autophosphorylation of the EGF
receptor leading to the formation of a multiprotein complex containing
the phosphorylated receptor, the phosphoprotein Shc, the adaptor
protein Grb2, and the Ras-GTP exchange factor
Sos(7, 8, 9) . Sos catalyzes exchange of GTP
for GDP on the small guanine nucleotide-binding protein, Ras, thereby
stimulating Ras activation(10) . Ras-GTP activates a kinase
cascade involving Raf, MEK, and MAP
kinase(11, 12, 13) . Activated MAP kinases
phosphorylate and activate transcription factors involved in cell
growth and proliferation(1) .-, G
-, G
-, and
G
-coupled receptors to stimulate MAP kinase activation have
also been assessed and
compared(14, 15, 16, 17, 18) .
In many cell types, G-coupled receptors mediate MAP kinase
activation via the -dependent activation of Ras (14, 15, 16) . Several of the intermediate
steps in the G-stimulated MAP kinase pathway are identical
with the RTK-stimulated signaling cascade including Shc
phosphorylation, Shc/Grb2 association, and Sos activation(19) .
Inhibitors of Src family tyrosine kinase activity abrogate
G-coupled receptor- and G-mediated Shc
phosphorylation and MAP kinase activation in COS-7 cells (3, 17, 20, 21) suggesting that a
Src family tyrosine kinase may be involved in the G-mediated
MAP kinase activation pathway at a point upstream of Ras activation.
The mechanism by which G subunits mediate activation of a
tyrosine kinase resulting in increased Shc phosphorylation, however, is
unclear.-mediated MAP kinase signaling pathway using two inhibitors
of PI-3K activity, wortmannin (30) and LY294002, and a dominant
negative mutant of the p85 subunit of PI-3K (
p85).
Materials
COS-7 and CHO cells were from the
American Type Culture Collection. Culture media and
LipofectAMINE
were from Life Technologies, Inc. Fetal
bovine serum (FBS) and gentamicin were from Life Technologies Inc.
LY294002 was from Bio-Mol. Monoclonal antibody 12CA5 was from
Boehringer Mannheim, and anti-ERK2 polyclonal antibody was from Santa
Cruz Biotechnology. Protein A-agarose was from Pharmacia Biotech Inc.
[-
P]ATP was from DuPont NEN. Myelin basic
protein (MBP) and wortmannin were from Sigma. UK-14304 was from Pfizer.
The cDNAs for the human 
2-C10 AR and ARK1 were cloned in our
laboratory(31) ; cDNAs encoding G1 and G2 were
provided by M. Simon; cDNA encoding hemagglutinin (HA)-tagged
p44
(ERK1) was from J. Pouysségur;
cDNA encoding p85 was from M. Sakaue. Constitutively active MEK
was from R. Erickson.Cell Culture and Transfection
COS-7 and CHO-K1
cells were maintained in Dulbecco's Modified Eagle's Medium
(DMEM) and F-12 medium, respectively, supplemented with 10% FBS and 50
µg/ml gentamicin. Cells were transiently transfected using
LipofectAMINE as described previously(17) . Assays were
performed 48 h after transfection. In MAP kinase activation assays,
transfected cells were serum-starved in DMEM containing 0.5% serum for
16 to 20 h prior to simulation.Measurement of MAP Kinase Activity
Activity of
epitope-tagged p44
(HA-ERK1), or
endogenous ERK2, was determined following immunoprecipitation, using
MBP as substrate (32) with modifications as
described(33) . Quantitation of labeled MBP was performed using
a Molecular Dynamics PhosphorImager.Measurement of Inositol Phosphate
Production
Transfected cells were labeled overnight with
[
H]inositol (1-2 µCi/ml) in DMEM
containing 10% FBS, washed once with phosphate-buffered saline, and
incubated in phosphate-buffered saline containing 1.0 mM CaCl
, 10 mM LiCl, and the indicated agonist
for 45 min. Cells were lysed in 0.4 M perchloric acid (1
ml/well), and 0.8-ml aliquots were neutralized with 0.4 ml of 0.72 M KOH, 0.6 M KHCO. Total inositol
phosphate accumulation was quantitated using Dowex anion exchange
chromatography as described previously(34) .Measurement of Ras Activation
Ras activation was
determined as described previously(14, 35) . Ras-bound
GDP and GTP were quantitated using a Molecular Dynamic PhosphorImager.
Ras activation is expressed as the amount of GTP bound to Ras as a
percent of total guanine nucleotide bound to Ras.
subunit of G mediates
Ras-dependent MAP kinase activation produced by stimulation of both the
lysophosphatidic acid (LPA) receptor and 
adrenergic
receptor (AR)(14) . In order to determine
whether PI-3K activity is required in the G-mediated
mitogenic signaling pathway, we assessed the effect of two chemical
PI-3K inhibitors (wortmannin and LY294002) and the effect of
overexpression of a dominant negative mutant of the p85 subunit of
PI-3K (p85) on G-coupled receptor- and
G-mediated MAP kinase activation. As shown in Fig. 1A, pretreatment of COS-7 cells with wortmannin or
LY294002 markedly inhibits MAP kinase activation produced by
stimulation of the endogenously expressed LPA receptor. This inhibition
of the LPA signal was also detectable at the level of endogenous MAP
kinase (ERK2) (Fig. 1A, right panel). Similar
inhibition by wortmannin and LY294002 is observed on MAP kinase
activation provoked by stimulation of transiently overexpressed
AR and direct transfection of G (Fig. 1B). Wortmannin and Ly294002 pretreatment also
inhibit GPCR- and G-mediated MAP kinase
activation in CHO-K1 cells (data not shown). In contrast, wortmannin
and LY294002 have a lesser effect on MAP kinase activation stimulated
by phorbol 12-myristate 13-acetate (PMA), or epidermal growth factor
(EGF) (Fig. 1B). The inhibition by wortmannin and
LY294002 is limited to the MAP kinase signaling pathway, in that the
PI-3K inhibitors do not affect AR- or
G-mediated phosphoinositide hydrolysis (Fig. 1C). Both wortmannin and LY294002 inhibit LPA and
G-stimulated MAP kinase activation in a
concentration-dependent manner (Fig. 1, D and E), with IC
values of 100 nM and 1.0
µM for wortmannin and LY294002, respectively.
-coupled receptor- and G-mediated MAP kinase
activation and IP production. COS-7 cells were cotransfected with
plasmid DNA encoding p44
(0.1
µg/well) and either pRK5 alone (2.0 µg/well), G1 and
2 (1.0 µg each/well), or AR (0.2
µg/well). Cells in A (right panel) were
untransfected, stimulated with 10 µM LPA, and assayed for
endogenous ERK2 activity. Cells were pretreated for 15 min with
wortmannin (1.0 µM), LY294002 (20 µM), or
vehicle (A, B, and C) or with the indicated
concentration of wortmannin (D) or LY294002 (E).
Cells were stimulated with LPA (10 µM), the
AR agonist UK-14304 (10 µM), or PMA (1.0
µM) unless indicated for 5 min (A, B, D, and E), and MAP kinase activation was determined
for 45 min (C) and IP production was measured. In A, B, and C, data are expressed as fold stimulation
where basal is defined as 1.0. In D and E, data are
expressed as a percent of the stimulation produced by LPA or
G in the absence of wortmannin or LY294002. Values in A are the mean ± S.D. from one representative experiment. All
other values are the mean ± S.E. from at least three separate
experiments. The absence of error bars indicates the S.D. or S.E. is
smaller than the size of the symbol.
(p85), which lacks the p110 binding site,
inhibits insulin-stimulated PI-3K activity and PIP production. The effect of p85 expression on
G-coupled receptor- and G-stimulated MAP kinase
activation is shown in Fig. 2. Expression of p85 inhibits
LPA-, AR-, and G-stimulated MAP kinase
activation without affecting MAP kinase activation stimulated by PMA.
Thus, expression of a dominant negative mutant of PI-3K produces
effects similar to wortmannin and LY294002. These results strongly
suggest that PI-3K activity is an essential component of the
G-mediated MAP kinase signaling pathway.
p85 expression on
G-coupled receptor- and G-mediated MAP kinase
(HA-ERK1) activation. COS-7 cells were cotransfected with plasmid DNA
encoding p44 (0.1 µg/well)
and either pRK5 alone (2.0 µg/well), G1 and G2 (1.0
µg each/well), or AR (0.2 µg/well) or pRK5
(2.0). Cells were also cotransfected with p85 (2.0 µg/well)
where indicated. Cells were stimulated with vehicle, LPA (10
µM), the AR agonist UK-14304 (10
µM), PMA (1.0 µM), or EGF (10 ng/ml) for 5
min, and MAP kinase activation was determined. The data are expressed
as fold stimulation where basal MAP kinase activity is defined as 1.0.
Values are mean ± S.E. from three separate
experiments.
-coupled receptor/G-mediated MAP kinase
signaling pathway precedes or follows Ras activation, the effect of
PI-3K inhibitors on G-coupled receptor-mediated Ras
activation was determined. As shown in Fig. 3, LPA-stimulated
Ras activation is abolished by wortmannin and LY294002 pretreatment. In
contrast, EGF-stimulated Ras activation is not significantly affected.
The striking sensitivity of the LPA-stimulated signal to inhibition of
PI-3K activity suggests a crucial role for PI-3K activity early in the
G-coupled receptor/G-mediated MAP kinase
signaling pathway.
-mediated MAP kinase signaling pathway is
provided by the results in Fig. 4. The effect of PI-3K
inhibition on MAP kinase activation provoked by overexpression of Sos,
constitutively active Ras (T24Ras), and constitutively active MEK
(MEK+) in COS-7 cells was assessed. Pretreatment with wortmannin
or LY294002 (Fig. 4A) or overexpression of p85
(data not shown) has no effect on the increase in MAP kinase activation
stimulated by Sos, T24Ras, or MEK+ (Fig. 4A),
suggesting PI-3K activity is upstream of these intermediates in the
G-mediated MAP kinase activation pathway.
-mediated mitogenic signaling pathway. COS-7 (A)
and CHO-K1 (B) cells were cotransfected with plasmid DNA
encoding p44 (0.1 µg/well)
and 1.0 µg/well of Sos, T24Ras, or MEK+ (A) and
G1/G2, Sos, or G1/G2 and Sos (B). Cells
were pretreated for 15 min with vehicle, wortmannin (1.0
µM), or LY294002 (20 µM), and MAP kinase
activity was determined. The data are expressed as fold MAP kinase
activity in which the basal MAP kinase activity is defined as 1.0.
Values are the mean ± S.E. from at least three separate
experiments.
with Sos results in a synergistic increase
in MAP kinase activation(19) . Expression of G or Sos
alone stimulates a 2-3-fold and 5-fold increase, respectively, in
MAP kinase activation (Fig. 4B). Coexpression of
G and Sos results in a 15-20-fold increase in MAP
kinase activation. As in COS-7 cells, G-mediated MAP kinase
activation in CHO-K1 cells is abolished by wortmannin or LY294002
pretreatment, and Sos-stimulated MAP kinase activation is unaffected.
The synergistic increase in MAP kinase activation produced by
G and Sos coexpression is not observed in cells pretreated
with wortmannin or LY294002 (Fig. 4B). Thus, the
ability of G subunits to synergize with Sos is dependent on
PI-3K activity, suggesting that PI-3K activity is required downstream
of G, but upstream of Sos in the G-mediated MAP
kinase activation signaling pathway. production(25, 26, 27, 28, 29, 43) .
Increased PI-3K activity is observed in anti-phosphotyrosine
immunoprecipitates following activation of G protein-mediated
systems(27) . Wortmannin attenuates platelet-activating
factor-stimulated MAP kinase activation in guinea pig neutrophils (24) and pertussis toxin-sensitive somatostatin
receptor-stimulated MAP kinase activation in CHO-K1 cells(22) .
The results of the present study demonstrate that PI-3K activity is
required in the G-mediated MAP kinase signaling pathway and
that the site of PI-3K activity in the pathway is upstream of Sos. -mediated Shc
phosphorylation is sensitive to tyrosine kinase inhibitors and
wortmannin(20) . Wortmannin-sensitive G-stimulated
PI-3K activity has been described in platelets and
neutrophils(44, 45, 46) , and a
G-sensitive PI-3K (designated p110 or PI-3K) has
been cloned(47) . It is thus attractive to speculate that
PI-3K activity may be required for recruitment and activation of
the tyrosine kinase(s) responsible for mediating Ras and MAP kinase
activation.),
is capable of binding with high affinity to the SH2 domains of proteins
such as Src and the p85 subunit of PI-3K(48) . Further,
PIP can compete with tyrosine-phosphorylated proteins for
binding to these sites. The PIP/SH2 interaction may suggest
a novel mechanism for regulating signaling pathways in PI-3K-dependent
systems. PIP may block phosphoprotein binding to SH2
domain-containing proteins or even supplant phosphoproteins bound to an
SH2 domain. It is therefore possible that PIP may serve as
an intermediate in the G-mediated MAP kinase signaling
pathway.p85 to inhibit G-mediated MAP
kinase activation may indicate a requirement for the p85-p110
complex in the pathway, possibly as part of a complex containing Shc
and a Src family tyrosine kinase(49) . Alternatively, p85
may inhibit the G signal by binding directly to
PIP. Binding of p85 to PIP may disrupt
PI-3K-dependent signaling by preventing PIP from competing
with phosphoproteins for binding to an SH2 domain. Therefore, it is
possible that both p85-p110 PI-3K- and PI-3K-dependent signaling
can be inhibited by expression of p85. Further investigation is
required to determine which PI-3K isotype is utilized in the
G-mediated MAP kinase signaling pathway, the mechanism by
which PI-3K activity provokes tyrosine kinase activation resulting in
Shc phosphorylation and the identity of the tyrosine kinase utilized in
this pathway.
), the subunit of the G
protein; MBP, myelin basic protein; DMEM, Dulbecco's modified
Eagle's medium; LPA, lysophosphatidic acid; PMA, phorbol
12-myristate 13-acetate; FBS, fetal bovine serum; AR,
adrenergic receptor; T24Ras, a constitutively active
mutant of Ras; p85, a dominant negative mutant of the p85 subunit
of PI-3K; PIP, phosphatidylinositol 3,4,5-trisphosphate;
SH2 and SH3, Src homology domains.
We thank Drs. W. J. Koch and J. R. Raymond for helpful
discussions and D. Addison and M. Holben for excellent secretarial
assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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