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Volume 272, Number 49, Issue of December 5, 1997
pp. 30599-30602
(Received for publication, June 10, 1997, and in revised form, August 27, 1997)
From the Friedrich Miescher Institute, CH-4002 Basel,
Switzerland
ABSTRACT We investigated the activation of the Ras/ERK
signaling pathway by 12-O-tetradecanoylphorbol-13-acetate
(TPA) in NIH3T3 fibroblasts. Interestingly, the activation was
suppressed not only by dominant negative Raf-1 but also by dominant
negative Ras and SOS. Further analysis revealed that TPA treatment
induced, dependently on protein kinase C, the mobility shift of
p66shc in SDS-polyacrylamide gel electrophoresis, which could
be prevented by treatment of the Shc immunoprecipitate with
serine/threonine-specific protein phosphatase 1 (PP1) or 2A (PP2A).
Phosphoamino acid analysis of Shc showed that unlike growth
factor-induced Shc phosphorylation, where Shc is mainly phosphorylated
at tyrosine residues, TPA-induced phosphorylation was only at serine
residues. Like growth factor-induced Shc phosphorylation, which leads
to the association of Shc with Grb2, TPA also induced this association,
but, correspondingly to the above results, the TPA-induced association
was disrupted by in vitro treatment of the Shc
immunoprecipitate with PP1. Taken together, these results suggest that
the TPA signal was fed at or upstream of Shc to activate the Ras/ERK
signaling pathway involving serine phosphorylation of Shc.
INTRODUCTION Ligand-activated growth factor receptors induce translocation of
the Grb2-SOS complex to the plasma membrane where p21ras is
localized by two alternative mechanisms: directly by recruiting Grb2
via its SH2 domain (1) or indirectly by recruiting Shc via its SH2 or
PTB domain (2), the subsequent tyrosine phosphorylation of which leads
to its association with Grb2 via its SH2 domain (3). The high affinity
binding of Grb2 to Shc proteins requires phosphorylation of Shc at
Tyr317, which lies within the high affinity binding motif
for the Grb2 SH2 domain (4).
12-O-Tetradecanoylphorbol-13-acetate
(TPA)1 is one of the
compounds widely used to probe various cellular activities. Its only known function in the cell is to activate protein kinase C (PKC), mimicking the physiological lipid metabolite diacylglycerol (5). Numerous studies have indicated that TPA activates the Ras/ERK signaling pathway (6-8), but it is still controversial as for the
entry site of the TPA signal in the pathway. In NIH3T3 fibroblasts protein kinase C phosphorylates Raf-1 and stimulates its kinase activity (9); however Raf-1 phosphorylation by PKC in vitro does not lead to MEK activation (10). It has also been reported that
the phosphorylation of Raf-1 is irrelevant to signal transduction (11).
We analyzed TPA-induced Ras/ERK signaling in the context of
ERK-mediated gene regulation in NIH3T3 cells and found that the TPA
signal is fed upstream of SOS and involves serine phosphorylation of
Shc.
MATERIALS AND METHODS The reporter plasmid pGL2 muPA-8.2 was constructed
by inserting the murine urokinase-type plasminogen activator (uPA) gene promoter (from NIH3T3 cells (0.1 × 106/well) were
plated in 6-well (35-mm) tissue culture plates with 2 ml DMEM
containing 10% CS and transfected 20 h later by the calcium
phosphate precipitation method (Pharmacia Biotech Inc.). Luciferase
expression was determined as described (13).
NIH3T3 cells (1 × 106) were plated in 10-cm dishes and transfected 20 h
later using 60 µl of LipofectAMINETM (Life Technologies,
Inc.) with 15 µg of pcDNA-p44-tag encoding HA-tagged ERK-1
together with 15 µg of the coexpressed plasmid for 5 h. The
cells were incubated in fresh DMEM with 10% CS for 5 h and in
DMEM with 0.1% CS for 12 h. The cells were then treated with
FGF-2 or TPA for 10 min and washed with phosphate-buffered saline.
Whole cell extracts were immunoprecipitated with anti-HA-tag mouse
monoclonal antibody (12CA5), and ERK activity was determined as
described (15).
NIH3T3 cells
stimulated for 10 min by TPA, FGF-2, or PDGF were immunoprecipitated as
described (16) using a polyclonal anti-Shc antibody (Transduction
Laboratories). The immunoprecipitates were analyzed by Western blots
using a monoclonal anti-Shc antibody (1:250; Transduction Laboratories)
or a monoclonal anti-Grb2 antibody (1:500; Transduction Laboratories).
An enhanced chemiluminescence detection method (Amersham) was employed,
and the membrane was exposed to Kodak X-Omat AR film.
NIH3T3 cells were grown to
confluency on a 15-cm dish, starved for 16 h in phosphate-free
DMEM containing 0.1% dialyzed calf serum, incubated for 4 h in
the same medium with the addition of 2 mCi of
[32P]orthophosphate, and then induced with 100 ng/ml TPA
or 10 ng/ml FGF-2 for 10 min. Cell extracts were immunoprecipitated
using polyclonal anti-Shc antibody, and the precipitates were
fractionated by SDS-polyacrylamide gel electrophoresis. Each Shc
isoform was recovered separately from the gel after autoradiography and
subjected to phosphoamino acid analysis as described (17).
RESULTS AND DISCUSSION In NIH3T3 cells, the uPA gene is activated by TPA and FGF-2
via the Ras/ERK signaling pathway (12). To know where in the pathway
the TPA signal is fed, we first examined the effects of various
signaling molecules on uPA gene induction in transient transfection
assays. The induction of the uPA promoter by TPA and FGF-2 was
suppressed by dominant negative mutants of Ras (Ras17N) or Raf-1
(N
[View Larger Version of this Image (28K GIF file)]
A constitutively active mutant of PKC It has been shown that PKC Both
[View Larger Version of this Image (32K GIF file)]
Translocation of the Grb2-SOS complex to the membrane is
induced either by Shc or by a receptor tyrosine kinase. Shc activation involves its phosphorylation at a tyrosine residue and subsequently interaction with Grb2 (22). Accordingly, both FGF-2 and PDGF, but not
TPA, induced tyrosine phosphorylation of all three Shc isoforms (Fig.
3A). Interestingly, all three
stimuli, TPA, FGF-2, and PDGF, led to a mobility shift of the
p66shc isoform (Fig. 3B), suggesting an alternative
modification on the Shc protein other than tyrosine phosphorylation
after induction with these agents.
[View Larger Version of this Image (53K GIF file)]
To analyze the mobility shift of the
p66shc isoform, Shc was immunoprecipitated from cell extracts
after stimulation of the cells with FGF-2 or TPA. The
immunoprecipitates were treated with either calf intestine alkaline
phosphatase (CIP), tyrosine-specific phosphatase LAR (23), or
serine/threonine-specific protein phosphatase 2A (24) (PP2A). Treatment
with either CIP or PP2A, but not LAR, prevented the mobility shift of
p66shc (Fig. 3C), suggesting that the shift is due
to serine/threonine phosphorylation. TPA-induced mobility shift of
p66shc was also sensitive to serine/threonine-specific protein
phosphatase 1 (PP1) (data not shown). The results in Fig. 3D
show that the mobility shift of p66shc induced by TPA is
dependent on protein kinase C; treatment of cells with the protein
kinase C inhibitor bisindolylmaleimide (25) or TPA for 24 h
down-regulating PKC prior to TPA treatment ablated the shift. The shift
induced by FGF-2 was partially suppressed by both treatments,
suggesting that serine/threonine phosphorylation of Shc after FGF-2
treatment also involves PKC isoforms, at least partially. Phosphoamino
acid analysis revealed that TPA and FGF-2 induced phosphorylation of
serine residues in p52shc and p66shc (Fig.
3E). Apparently, serine phosphorylation of p52shc
does not affect its mobility in SDS-polyacrylamide gel electrophoresis under the conditions used. In accordance with the result shown in Fig.
3A, phosphorylation of tyrosine residues was induced by FGF-2 but not by TPA.
We were
interested in whether the TPA-induced serine phosphorylation of Shc
also promotes its association with Grb2. After immunoprecipitation of
Shc from either TPA- or FGF-2-treated or untreated cells we could
detect an increase in Grb2 association with Shc after 2 min of either
treatment, which remained constant at least up to 30 min. The
association between Shc and Grb2 shows a similar duration as the
modification of the p66shc. In accordance with the results
described in Fig. 3, tyrosine phosphorylation of Shc was increased only
after FGF-2 treatment (Fig.
4A). When Shc
immunoprecipitates were treated with PP1, Grb2 association induced by
TPA or FGF-2 was suppressed or strongly reduced, respectively (Fig.
4B). The PP1-specific inhibitor calyculin A suppressed the
inhibitory effect of PP1.
[View Larger Version of this Image (48K GIF file)]
It has been well established that the major modification of Shc is the
phosphorylation of Tyr317, which is induced by many
receptor tyrosine kinases and cytoplasmic tyrosine kinases (Ref. 26 and
references cited therein). Tyrosine-phosphorylated Shc recruits the
Grb2-SOS complex to the membrane via the SH2 domain of Grb2, thus
allowing SOS to activate Ras. The present results suggest that Shc can
also recruit Grb2 through serine phosphorylation. An increase in serine
phosphorylation of Shc as an G FOOTNOTES ACKNOWLEDGEMENTS We thank Kurt-Ballmer Hofer, Nancy Hynes,
Patrick King, and Ruedi Meili for critical reading of the manuscript,
Bettina Moser for excellent assistance in phosphoamino acid analysis,
and Brian Hemmings for providing us with PP2A.
REFERENCES
COMMUNICATION:
12-O-Tetradecanoylphorbol-13acetate Activates
the Ras/ Extracellular Signal-regulated Kinase (ERK) Signaling
Pathway Upstream of SOS Involving Serine Phosphorylation of Shc in
NIH3T3 Cells*
,§,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
8, 200 to +398 with respect to the transcription initiation site) upstream of the luciferase-coding region of the promoter-less plasmid pGL2-basic (Promega). Expression vectors for
various signaling molecules used in this work were previously described
(12, 13) except that for constitutively active pPKC
(14).
Is Dependent on
SOS
Raf-1) or by mitogen-activated protein kinase-specific protein
phosphatase MKP-1 (Fig. 1A),
confirming the involvement of the Ras/ERK pathway in uPA gene
activation by TPA and FGF-2. Surprisingly, the induction by TPA was
also suppressed by dominant negative mutants of SOS (mSOS1, Fig.
1B), showing that TPA activates the Ras/ERK signaling
pathway at or upstream of SOS. Wild-type SOS (mSOS1) enhanced both the
basal and induced activities of the uPA promoter (Fig. 1B).
SOS is a GTP-GDP exchange factor which is able to activate Ras (18).
Accordingly, the induction of the uPA gene by constitutively active Ras
or Raf was not suppressed by coexpression of mSOS1 (data not shown),
indicating that inhibitory effects of dominant SOS are not general on
gene expression or protein synthesis.
Fig. 1.
Effects of dominant negative forms of various
signaling molecules on the activation of the uPA gene promoter by FGF-2
and TPA and by overexpression of constitutively active PKC in NIH3T3 cells. A, transient transfection with 1 µg of pGL2muPA-8.2 together with 0.3 µg of pMuLV-Ha-Ras (wt H-Ras), pRSV-H17
(mt Ras17N, dominant negative Ha-Ras), pc-c-Raf (wt Raf-1),
pCMV-Nraf (mt NRaf-1, dominant negative c-Raf-1), or
pSG5-3CH134 (wt MKP-1, wild-type MAP kinase phosphatase).
Cells were treated without (open bars) or with 10 ng/ml
FGF-2 (solid bars) or 100 ng/ml TPA (gray bars)
for 8 h. B, transient transfection with 1 µg of
pGL2muPA-8.2 together with 0.3 µg of pSR
-mSOS1 (wt
mSOS1) and pSR-mSOS1 (mt mSOS1, dominant
negative). Cells were treated as above. C, transient transfection with 1 µg of pGL2muPA-8.2 together with 0.3 µg of several expression vectors as above and either with (solid
bars) or without (open bars) 0.3 µg of pPKC RA, an
expression vector for constitutively active pPKC.
(PKC RA) strongly activated
the uPA promoter in the transient transfection assay, but the
activation was not blocked by mSOS1 (Fig. 1C). NRaf-1 and wild-type MKP-1 were able to block this induction. These results show that PKC RA activates the pathway at a step downstream of SOS.
phosphorylates Raf-1 at the same sites in
a cell-free system which are phosphorylated in vivo (9).
Accordingly, overexpressed PKC may activate Raf-1 directly in the
cytoplasm without Ras-dependent translocation of Raf-1 to
the membrane (19), possibly because of an increase in the number of
PKC molecules in the transfection assays described here. In the case
of TPA induction, which activates several PKC isoforms including and (20), the absolute number of PKC molecules is not elevated and,
therefore, Ras-dependent translocation of Raf-1 to the
membrane may still be needed for the induction.
mSOS1 and Ras17N
strongly suppressed not only the uPA promoter activity but also the
ERK-1 activity induced by TPA and FGF-2 (Fig.
2A). mSOS1 showed no
inhibitory effect on ERK-1 activity, and constitutively active Ras
(Ras61L) alone activated ERK-1 as strongly as TPA or FGF-2. The levels
of tagged-ERK-1 expression were similar in different transfections
(Fig. 2B). Pretreatment of the cells for 18 h with an
inhibitor of farnesylation, an obligatory step in Ras processing (21),
blocked TPA and FGF-2 stimulation of ERK-1 activity about 60% in both
inductions (Fig. 2C), proving again that the activation of
Ras is necessary for activation of the ERK pathway by TPA and
FGF-2.
Fig. 2.
Effects of mSOS1 and Ras17N and of a Ras
inhibitor on ERK-1 activity induced by TPA and FGF-2. A,
cells were transfected with p44mapk-tag alone (bars
1-3) or together with SRSOS1 (bars 4-6),
SRSOS1 (bars 7-9), pRSV H17 (bars 10-12), or
pRSV61L (constitutively active Ha-Ras, bar 13) and were
uninduced (open bars) or induced by 100 ng/ml TPA
(solid bars) or 10 ng/ml FGF-2 (gray bars) for 10 min. ERK-1 kinase activity was measured using myelin basic protein as
substrate. B, levels of HA-tagged ERK-1 expression analyzed
by Western blotting. Lane numbers correspond to bar
numbers of A. C, cells were either untreated
(open bars) or pretreated (solid bars) with 50 µM FPT inhibitor III (Calbiochem) for 18 h and then
stimulated for 10 min with TPA or FGF-2.
Fig. 3.
Modification of Shc. A, tyrosine
phosphorylation of Shc. Cells were induced by treatment for 10 min with
100 ng/ml TPA, 10 ng/ml FGF-2, or 30 ng/ml PDGF and immunoprecipitated
with the Shc antibody. Immunoblotting was performed using a mouse
monoclonal anti-phosphotyrosine antibody. B, retardation of
the p66shc isoform in SDS-gel electrophoresis. The same blot as
in A was washed and reprobed using a mouse monoclonal
anti-Shc antibody. C, effects of various phosphatases on the
mobility shift of Shc. Shc immunoprecipitates were treated with either
calf intestine alkaline phosphatase (Boehringer Mannheim) (10 units/sample) at 37 °C or specific tyrosine phosphatase LAR (New
England Biolabs) (10 units/sample) or PP2A (provided by Brian Hemmings)
(10 nM) at 30 °C for 2 h, then immunoblotted with a
mouse monoclonal anti-Shc antibody. D, effects of PKC
inactivation on the TPA- and FGF-2-induced p66shc mobility
shift. Cells were pretreated with 500 nM PKC inhibitor (bisindolylmaleimide; Calbiochem) for 1 h or 100 ng/ml TPA for 24 h to down-regulate PKC before stimulation with TPA or FGF-2 for
10 min. Shc immunoprecipitates were analyzed by Western blotting using
a mouse monoclonal anti-Shc antibody. E, phosphoamino acid analysis. Cells were induced with TPA or FGF-2 in the presence of
radioactive orthophosphate. Shc immunoprecipitates were fractionated, and each isoform was separately analyzed as described under
"Materials and Methods." Serine phosphorylation of p52shc
was stimulated 5- and 1.5-fold, while that of p66shc was
stimulated 3- and 2-fold by TPA and FGF-2, respectively.
Fig. 4.
TPA-induced Shc association with Grb2.
A, cells were induced with 100 ng/ml TPA or 10 ng/ml FGF-2
for the different times indicated, and cell extracts were
immunoprecipitated using a polyclonal anti-Shc antibody and analyzed by
Western blotting using a rabbit polyclonal anti-Shc antibody. The same
blot was reprobed with either anti-phosphotyrosine antibody (4G10) or
mouse monoclonal anti-Grb2 antibody. B, Shc
immunoprecipitates from control (), TPA-induced, or FGF-2-induced
cells were treated at 30 °C for 30 min with PP1 (Calbiochem; 5 units), PP1 together with calyculin A (50 nM), or calyculin
A alone and analyzed by Western blotting using mouse monoclonal anti-
Grb2 antibody.
-induced event (27) or in response
to epidermal growth factor stimulation (28) has been observed, but its
biological significance has not been addressed. It has been shown
recently that serine phosphorylation of Raf-1 is involved its
interaction with 14-3-3 protein (29) and that serine phosphorylation of Raf-1 is also involved in the recruitment of the Fyn SH2 domain (30),
although it is not known whether this transduces signaling. Taken
together, our results suggest that TPA can activate Ras/ERK signaling
by inducing serine phosphorylation of Shc. It remains to be seen
whether PKC phosphorylates Shc directly or indirectly and how the
serine phosphorylation of Shc is able to promote the interaction of Shc
with Grb2.
The first two authors contributed to the work equally.
§
Present address: The Rockefeller University, Dept. of Molecular
Oncology, 1230 York Ave., New York, NY 10021.
¶
To whom correspondence should be addressed. Tel.:
41-61-6976669; Fax: 41-61-6973976; E-mail: nagamine{at}fmi.ch.
1
The abbreviations used are: TPA,
12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C;
uPA, urokinase-type plasminogen activator; DMEM, Dulbecco's modified
Eagle's medium; CIP, calf intestine alkaline phosphatase; PP1, protein
phosphatase 1; PP2A, protein phosphatase 2A; ERK, Ras/extracellular
signal-regulated kinase; CS, calf serum; HA, hemagglutinin; FGF,
fibroblast growth factor; PDGF, platelet-derived growth factor.
Volume 272, Number 49,
Issue of December 5, 1997
pp. 30599-30602
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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