Regulation of RAS in human platelets
Evidence that activation of RAS is not sufficient to lead to ERK1-2 phosphorylation
David Tulasne
1
, Teresa Bori
2
and Steve P. Watson
1
1
Department of Pharmacology, University of Oxford, UK;
2
Division of Medical Sciences, The Medical School,
University of Birmingham, Edgbaston, UK
In this study, w e show that the G protein-coupled receptor
agonist thrombin, the glycoprotein VI agonist convulxin,
and the cytokine receptor Mpl agonist thrombopoietin
(TPO) are able to induce activation of RAS in human
platelets. Recr uitment o f G RB2 by tyrosine-phosphorylated
proteins in response to TPO and convulxin but not by
thrombin occurred with a s imilar time-course to RAS a cti-
vation, consistent with a causal relationship. On the other
hand, activation o f ERK2 b y thrombin a nd convulxin i s
delayed and also inhibited by t he protein kinase C inhibitor
Ro-31 8220, whereas R AS activation is unaffected. Further
evidence for differential regulation of RAS and ERK is
provided by the observations that TPO, which activates
RAS but not p rotein kinase C, does not activate ERK, and
that th e i nhibitor of SRC kinases PP1 inhibits activation of
RAS but not ERK2 in response to thrombin. Our results
demonstrate that activation of RAS is not necessarily cou-
pled to ERK i n human platelets.

and thrombin stimulate GDP–GTP exchange of RAS [5];
the cytokine receptor Mpl agonist thrombopoietin (TPO) is
able to induce activation of RAF [6]; and collagen and
thrombin induce MEK and ERK activation [7]. In the
present study, we show that whereas RAS is activated in
platelets in response to activation by thrombin, glycoprotein
VI (GPVI) agonists and TPO, this is not necessarily coupled
to activation of ERK.
MATERIALS AND METHODS
Antibodies and reagents
Anti-RAS (clone RAS 10, recognizing p21 H-, K- and
N-RAS) and anti-phosphotyrosine 4G10 monoclonal Ig
were purchased f rom Upstate Biotechnology (TCS Biologi-
cals Ltd, UK). Anti-SHC polyclonal and anti-GRB2 mono-
clonal Ig w ere purchased from Transduction Laboratories
(Becton Dickinson Ltd, UK). Anti-GRB2 polyclonal Ig and
anti ERK2 polyclonal Ig were purchased from Santa Cruz
Biotechnology, Inc. Anti-(phospho active ERK phospho-
Thr202/Tyr204) polyclonal Ig was purchased from Promega
Biosciences, Inc. Anti-SYK rabbit polyclonal serum was a
generous gift of M. Tomlinson (DNAX, Palo Alto, C a,
USA). Gluthatione S-transferase (GST)–RAF–RBD fusion
protein was generous gift of Dr F. McKenzie (CNRS UMR
134, Nice, France). Hu man r ecombinant TPO was from
Genentech, Inc. Other reagents were from previously
described sources [8,9].
Platelet preparation
Blood samples were collected from healthy volunteer donors
into 1/10 vol. 3.8% trisodium citrate (w/v) and then 1/10 vol.
of acid/citrate/dextrose ( ACD; 120 m

2
HPO
4
,2.9m
M
KCl, 12 m
M
NaHCO
3
,20m
M
Hepes,
5m
M
glucose, 1 m
M
MgCl
2
pH 7.3) and 3 mL ACD in the
presence of prostacyclin (0.1 lgÆmL
)1
). Platelets w ere recen-
trifuged at 1000 g for 10 min and resuspended at 5 · 10
8
plateletsÆmL
)1
in Tyrode’s/Hepes buffer. Platelet stimula-
tions were performed at 37 °C in a PAP4 aggregometer with
continuous stirring at 1200 r.p.m. (BioData Corporat ion).
Immunoprecipitation

leupeptin, 10 lgÆmL
)1
aprotinin,
1 lgÆmL
)1
pepstatin A, pH 7.3). Lysed cells and debris
were removed by centrifugation. Cell lysates were precleared
for 1 h at 4 °C with protein A–Sepharose. Platelet lysates
were incubated overnight at 4 °Cwith3lL anti-GRB2 or
anti-SHC polyclonal Ig under constant rotation. Protein A–
Sepharose was added and samples were rotated for a further
60 min. The pellet of protein A–sepharose was washed once
in lysis buffer and three times in NaCl/Tris/Tween (10 m
M
Tris, 160 m
M
NaCl, 0.1% Tween 20 (pH 7.3)].
GST precipitation
Platelets (4 · 10
8
cellsÆmL
)1
)treatedfor10minwith
2UÆmL
)1
apyrase, 10 l
M
indomethacin and 1 m
M
EGTA

Tris, 10 m
M
EDTA, 2 m
M
phenylmethylsulphonyl fluoride
,
10 lgÆmL
)1
leupeptin,
pH 7.3). Cell l ysates were centrif ugated at 4 °Cfor2.5h
at 100 000 g. Pellets containing cytoskeleton proteins were
solubilized in SDS sample buffer ( 40% glycerol, 8% SDS,
20% b-mercaptoethanol, 0.008% Bromophenol blue,
250 m
M
Tris/HCl pH 6.8). Proteins from supernatant and
Triton X-100 insoluble-pellet were resolved on S DS/PAGE
and t ransferred to PVDF membrane. Blots were developed
using the ECL system.
Platelet labelling with [
32
P]orthophosphate
Platelets suspended i n Tyrode’s/Hepes without phosphate
were incubated with [
32
P]orthophosphate (0.5 mCi ÆmL
)1
)
for 1 h at 37 °C. Platelets were washed once in Tyrode’s/
Hepes and resuspended at 5 · 10

)1
)werepre-
treated or n o t for 10 min with 10 m
M
EGTA or 1 m
M
of RGDS
peptide. Platelets were then stimulated with 1 UÆmL
)1
thrombin or
150 n g ÆmL
)1
TPO. Platelets were lysed by the addition of Triton X-100
lysis buffer a t 30, 60, 1 20 or 240 s. Triton X-100 so luble and insoluble
fractions were isolated. Proteins of both fractions were resolved by
12.5% SDS/PAGE and analysed by Western blotting using an
anti-RAS Ig.
1512 D. Tulasne et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Thombin, convulxin and TPO induce activation of RAS
RAS activation was measured through the ability of its
activated form (RAS–GTP) to bind to a GST fusion protein
consisting of the RAS-binding domain of RAF and
subsequent detection by Western blotting. To prevent trans-
location of RAS t o t he insoluble fraction a nd secondary
responses, aggregation mediated by t he integrin GPIIb-IIIa
was inhibited by EGTA and the activation m ediated by
thromboxane A
2
and ADP were blocked by indomethacin
and apyrase, respectively. Thrombin, convulxin and TPO

concentration-dependent manner with different time-course. (A, C and
E) Washed human platelets (4 · 10
8
ÆmL
)1
), prepared in buffer con-
taining EGTA, indometacine and apyrase, were stimulated with
increasing concentrations of thrombin for 120 s, convulxin f or 120 s
and TPO for 240 s. (B, D and F) Washed human platelets were sti-
mulated as indicated time with 1 UÆmL
)1
thrombin, 10 lgÆmL
)1
convulxin or 150 ngÆmL
)1
TPO. Platelets were then lysed b y the
addition of RAS lysis buffer. Cell extracts were preci pitated using GST
fusion protein con taining the RAS-b inding domain o f RAF. Precipi-
tated proteins were resolved by 12.5% SDS/PAGE and analysed by
Western blotting using an anti-RAS Ig (top). Proteins of whole cell
lysate were resolved by 12.5% SDS/PAGE and analysed by Western
blotting using anti-RAS Ig (bottom). Results p resented are represen-
tative of th ree experiments.
Fig. 3. Convulxin and TPO but not thrombin induce the recruitment of
GRB2 on pho sphorylated a dapters. ( A, B and C) W ashed human
platelets were stimulated with 1 UÆmL
)1
of thrombin for 120 s,
10 lgÆmL
)1

representative of three experiments.
Ó FEBS 2002 Regulation of RAS in platelets (Eur. J. Biochem. 269) 1513
phosphorylated protein was detected at 50 kDa. No change
in the pattern of phosphorylated proteins associated with
GRB2 was observe d in p latelets stimulated by thrombin.
Convulxin and TPO stimulated tyrosine phosphorylation of
the 50 kDa adapter S HC, l eading t o fo rmation o f a
complex with GRB2 (Fig. 3B). In convulxin-stimulated
platelets, GRB2 also coimmunoprecipitated with the a dapter
protein LAT and the tyrosine kinase SYK, suggesting that
these phosph orylated proteins could also recruit GRB2
after stimulation (Fig. 3C). Time-cou rse s tudies indicated
that SHC phosphorylation and GRB2 association in
response t o TP O o ccurred g rad ually with a maximal
response at 240 s (Fig. 3D). In convulxin stimulated-
platelets, association of phosphorylated proteins with
GRB2 was maximal at 10 s and was sustained for at least
240 s (Fig. 3E). The time-course of RAS activation in
response to convulxin and TPO was similar to the
recruitment of GRB2 to phosphorylated adapters.
RAS and ERK are regulated differently
The activation of ERK1 and 2, the downstream k inases of
the RAS–ERK signalling pathway, was measured by
Western blotting using an anti-(phospho-specific ERK1-2)
Ig. Thrombin and convulxin were able to induce phos-
phorylation of ERK kinase after a delay of 60 s (Fig. 4A
and B). Although we have p reviously shown weak phos-
phorylation of ERK1 in r esponse to thrombin and collagen
[7], only ERK2 phosphorylation was detected using the
antiphospho ERK1-2. In contrast, TPO was unable to

10minwith10l
M
Ro 31 8220 and stimulated for 2 min with
1UÆmL
)1
thrombin, for 2 min with 10 lgÆmL
)1
convulxin or f or
5 min with 150 ngÆmL
)1
TPO. (A) S timulated l abelled-platelets w ere
lysed in Laemmli sa mple buffer a nd were resolved by 12% SDS/
PAGE. Phosphorylated pleckstrin w as detected by autoradiography.
(B) Platelets were lysed by the addition of denaturating lysis buffer.
Proteins of whole c ell lys ate were r esolved by 1 0% S DS/PAGE a nd
analysed by We stern b lotting using an anti-(phospho-specific ERK1-2)
Ig (top). The filter w as stripped and reprobed using an anti-ERK2 I g
(bottom). (C) Platelets were lysed by the a ddition of RAS lysis buffer.
Cell extracts were precipitated using GST fusion protein containing
the RAS-binding domain of RAF. Precipitated proteins were resolved
by 12.5% SDS/PAGE and analysed by Western blotting using an
anti-RAS Ig (top). Proteins o f whole cell lysate were resolved by
12.5% SDS/PAGE and analysed by Western blotting using anti-RAS
Ig (bottom). Resu lts presented are rep resentative of three experi-
ments.
Fig. 4. Thrombin and convulxin but not TPO induce ERK activation.
(A, B and C) Washed human platelets were stimulated for the times
indicatedwith1UÆmL
)1
thrombin, 10 lgÆmL

linking of GPVI, which is associated with the Fc receptor c
chain. The GPVI-Fc receptor c-chain signalling pathway
shares many features with those of ITAM-containing
receptors in the immune system [13]. Following T-cell
receptor activation, recruitment of GRB2 by LAT and SHC
was s hown to be involved in the activation of the RAS-
ERK signalling pathway [14,15]. The time-course of RAS
activation in response to convulxin was similar to the
recruitment of GRB2 t o phosphorylated adapters, suggest-
ing a possible r egulation of RAS by convulxin through this
mechanism.
In response to TPO, an association between GRB2 and
phosphorylated SHC was identified. This interaction
occurred with a similar time-course to activation o f RAS,
suggesting a causal relationship. In megakaryocytic cell
lines, it has been shown that GRB2 recruitment by
phosphorylated SHC f ollowing c-Mpl r eceptor activation
by TPO contributes to activation of the RAS–ERK
signalling pathway [16].
The S RC kinase family inhibitor PP1 inhibited a ctiva-
tion of RAS in response to thrombin. Activation of RAS
by G coupled-receptors in oth er cell types is also mediated
through the SRC kinases [17–19]. PP1 also inhibited
activation of RAS stimulated by convulxin, consistent with
the r ole o f t hese kinases in m ediation of the phosphory-
lation of the ITAM motif of the Fc receptor c chain
[11,12].
As a principal mechanism, the activation of RAS leads to
the activation of ERK1-2 b y sequential a ctivation of R AS–
RAF–MEK and ERK. In platelets, phosphorylation of

PP1 and stim ulated for 2 min with 1 UÆmL
)1
thrombin, for 2 min with 10 lgÆmL
)1
convulxin or for 5 m in with
150 ngÆmL
)1
TPO. (A) Platelets were lysed b y the addition of denat-
urating lysis buffer. Proteins of whole cell lysate were resolved by 10%
SDS/PAGE and analysed by Western blotting using an anti-(phospho-
specific ERK1-2) Ig (top ). The filter was strippe d and reprobed using
an anti-ERK2 Ig (bottom). (B) Platelets were lysed by the a ddition of
RAS lysis buffer. Cell extracts were precipitated using GST fusion
protein c ontaining the RA S-binding domain of RAF. Precipitated
proteins were resolved by 12.5% SDS/PAGE and analysed by Western
blotting using an anti-RAS Ig (top). Proteins of whole cell lysate were
resolved by 12.5% SDS/PA GE and a nalysed by Western blotting
using anti-RAS Ig (bottom). Results presented are repr esentative of
three experiments.
Ó FEBS 2002 Regulation of RAS in platelets (Eur. J. Biochem. 269) 1515
Nevertheless, RAS could participate in the regulation of
ERK b y potentiating the activation mediated by PKC. For
instance, it has been shown that TPO is able to potentiate
ERK activation induced by thrombin [6]. The authors
proposed that this could be due to the a bility of TPO to
activate the e arly events of the R AS signalling pathway.
However, a recent study reported that inhibitors of
PtdIns3K abolished potentiation of ERK by TPO in
response to thr ombin, demonstrating that po tentiat ion is
mediated though the PtdIns3K pathway. The authors

over-expression of proteins responsible for down-regulation
could be an explanation for the inefficiency of RAS to
activate ERK in platelets.
RAS–RAF i nteraction and s ubsequent regulation of ERK
is not the only pathway regulated by RAS. For instance, a
mutated form of RAS that is unable to bind RAF is still
able to induce cytoskeletal rearrangements through activa-
tion of the small G protein RAC [30] and is able to regulate
PtdIns3K [31]. Relocalization of RAS from the cytoplasmic
to the cytoskeleton fraction could suggest an involvement
of RAS during cytoskeleton rearrangement of platelets.
Our study shows that in platelets RAS is not sufficient by
itself to induce activation of its main downstream t arget
ERK. Platelets appear to be a model with which to study
down-regulation of the RAS–ERK signalling p athway and
other functions of RAS. Down-regulation of the RAS–
ERK pathway may be a critical step in the process of end-
stage megakaryocyte differentiation.
ACKNOWLEDGEMENTS
This work was supported by the British Heart Foundation (BHF).
S. P. W. is a BHF Senior Research Fellow .
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