Watson1 1 University of Birmingham, Edgbaston, UK In this study, we show that the G protein-coupled receptor agonist thrombin, the glycoprotein VI agonist convulxin, and the cytokine rec
Trang 1Regulation of RAS in human platelets
Evidence that activation of RAS is not sufficient to lead to ERK1-2 phosphorylation David Tulasne1, Teresa Bori2and Steve P Watson1
1
University of Birmingham, Edgbaston, UK
In this study, we 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 Recruitment of GRB2 by tyrosine-phosphorylated
proteins in response to TPO and convulxin but not by
thrombin occurred with a similar time-course to RAS
acti-vation, consistent with a causal relationship On the other
hand, activation of ERK2 by thrombin and convulxin is
delayed and also inhibited by the protein kinase C inhibitor
Ro-31 8220, whereas RAS activation is unaffected Further
evidence for differential regulation of RAS and ERK is provided by the observations that TPO, which activates RAS but not protein kinase C, does not activate ERK, and that the inhibitor 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 in human platelets
Keywords: ERK; glycoprotein VI; platelet; protein kinase C RAS; signalling; thrombin; thrombopoietin
RAS is a ubiquitously expressed GTPase protein, which is
activated following conversion from a GDP to GTP-bound
state GDP–GTP exchange is stimulated by SOS, which is
constitutively associated through its proline rich domain to
the SH3 domains of the adapter GRB2 The Src homology
(SH)2 domain of GRB2 is able to bind phosphorylated
tyrosine residues on tyrosine kinase receptors or
membrane-localized adapters Localization of GRB2–SOS complex to
the plasma membrane, following this recruitment, promotes
RAS activation [1–3]
RAS was discovered as an oncogene, which was in its
activated state by constitutive binding of GTP A number of
proteins, including RAF, GAPp120, MEKK1, and
phos-phatidylinositol-3 kinase (PtdIns3K) are able to interact
with activated RAS (for review see [4]) The interaction of
RAS with the serine threonine kinase RAF is the most
thoroughly characterized of these interactions RAF
regu-lates the kinases MEK, which in turn activates ERK1-2
(MAPK p44, p42) ERK1-2 are able to regulate a number
of transcription factors, cytoplasmic proteins and
down-stream kinases
RAS and all of the components of the RAS–ERK
signalling pathway (RAS, RAF, MEK, ERK) are expressed
in platelets and undergo activation It has been shown that
the G protein-coupled receptor agonists thromboxane A2 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
M A T E R I A L S A N D M E T H O D S Antibodies and reagents
Anti-RAS (clone RAS 10, recognizing p21 H-, K- and N-RAS) and anti-phosphotyrosine 4G10 monoclonal Ig were purchased from Upstate Biotechnology (TCS Biologi-cals Ltd, UK) Anti-SHC polyclonal and anti-GRB2 mono-clonal Ig were 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, Ca, USA) Gluthatione S-transferase (GST)–RAF–RBD fusion protein was generous gift of Dr F McKenzie (CNRS UMR
134, Nice, France) Human recombinant 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 mM sodium citrate/
110 m glucose/80 m citric acid) was added Platelet-rich
Correspondence to D Tulasne, Department of Pharmacology,
University of Oxford, Mansfield Road, Oxford OX1 3QT, UK.
Fax: +44 1865 271853, Tel.: + 44 1865 271590,
E-mail: david.tulasne@pharmacology.oxford.ac.uk
Abbreviations: GST, gluthatione S-transferase; GPVI, glycoprotein
VI; PtdIns3K, phosphatidylinositol 3-kinase; PVDF, poly(vinylidene
difluoride); PKC, protein kinase C; PP1,
(4-amino-4-(4-methylphe-nyl)-7-(t-butyl)pyrazola[3,4-d]pyrimidine); SH2, Src homology 2;
SH3, Src homology 3; TPO, thrombopoietin.
(Received 19 October 2001, revised 1 November 2001, accepted
21 January 2002)
Trang 2plasma was obtained by centrifugation at 200 g for 20 min.
Platelets were isolated from platelet-rich plasma by
centrif-ugation at 1000 g for 10 min, in the presence of prostacyclin
(0.1 lgÆmL)1) The pellet was resuspended in 25 mL of a
modified Tyrode’s/Hepes buffer (134 mM NaCl, 0.34 mM
Na2HPO4, 2.9 mMKCl, 12 mMNaHCO3, 20 mMHepes,
5 mMglucose, 1 mMMgCl2pH 7.3) and 3 mL ACD in the
presence of prostacyclin (0.1 lgÆmL)1) Platelets were
recen-trifuged at 1000 g for 10 min and resuspended at 5· 108
plateletsÆmL)1in 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 Corporation)
Immunoprecipitation
Platelets (4· 108 cellsÆmL)1) treated for 10 min with
2 UÆmL)1apyrase, 10 lMindomethacin and 1 mMEGTA
were lysed with an equal volume of ice-cold Nonidet P-40
buffer [20 mMTris, 300 mMNaCl, 2 mMEDTA, 2% (v/v)
NP40, 1 mM phenylmethanesulfonyl fluoride, 2 mM
Na3VO4, 10 lgÆmL)1 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°C with 3 lL 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 mM
Tris, 160 mMNaCl, 0.1% Tween 20 (pH 7.3)]
GST precipitation
Platelets (4· 108 cellsÆmL)1) treated for 10 min with
2 UÆmL)1apyrase, 10 lMindomethacin and 1 mMEGTA
were lysed with an equal volume of ice-cold Nonidet P-40
buffer containing additional 1% N-octyl glucoside and
5 mM MgCl2 Lysed cells and debris were removed by
centrifugation Cell lysates were precleared for 1 h at 4°C
with glutathione–agarose Platelet lysates were incubated
3 h at 4°C with 5 lg GST–RAF–RBD immobilized on
agarose The pellet was washed once in lysis buffer and three
times in NaCl/Tris/Tween
Immunoblotting
For ERK phosphorylation, platelets were lysed in 10·
de-naturating buffer (10% SDS, 100 mMNaCl, 50 mMTris,
pH 7.3) Lysed cells and debris were removed by
centrifu-gation Laemmli buffer was added, and the lysate was boiled
for 2 min Proteins were separated by SDS/PAGE and
transferred to poly(vinylidene difluoride) (PVDF)
mem-brane Blots were developed by using the enhanced
chemi-luminescence detection (ECL) system
Cytoplasmic and cytoskeleton localization
Platelets were prepared and stimulated as described above
and then lysed with an equal volume of ice-cold Triton
X-100 lysis buffer (100 mM Tris, 10 mM EDTA, 2 mM
phenylmethylsulphonyl fluoride, 10 lgÆmL)1 leupeptin,
pH 7.3) Cell lysates were centrifugated at 4°C for 2.5 h
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 mMTris/HCl pH 6.8) Proteins from supernatant and Triton X-100 insoluble-pellet were resolved on SDS/PAGE and transferred to PVDF membrane Blots were developed using the ECL system
Platelet labelling with [32P]orthophosphate Platelets suspended in Tyrode’s/Hepes without phosphate were incubated with [32P]orthophosphate (0.5 mCiÆmL)1) for 1 h at 37°C Platelets were washed once in Tyrode’s/ Hepes and resuspended at 5· 108 plateletsÆmL)1 in Tyrode’s/Hepes plus indomethacin and left 15 min before the experiment as described above Reactions were stopped using Laemmli buffer Proteins were resolved on SDS/ PAGE and visualized by autoradiography
R E S U L T S Platelet aggregation induces RAS localization
to cytoskeleton Subcellular localization of RAS was determined after stimulation of platelets by thrombin, convulxin and TPO
In nonstimulated platelets, RAS was detected in cytoplas-mic and cytoskeletal fractions, corresponding, respectively,
to detergent soluble and insoluble fractions (Fig 1) Aggregation in response to thrombin induced translocation
of RAS from the cytoplasmic fraction to the cytoskeleton fraction (Fig 1A) In the presence of EGTA or RGDS peptide, both of which inhibit GPIIb-IIIa-dependent aggre-gation, relocalization of RAS was not observed (Fig 1B and C) A similar set of results was obtained in response to aggregation induced by the GPVI agonist, convulxin (data not shown) TPO does not induce aggregation or trans-location of RAS to the cytoskeletal fraction (Fig 1D)
Fig 1 Aggregation in response to thrombin and convulxin induces relocalization of RAS from the cytoplasmic to the cytoskeleton fraction (A, B, C and D) Washed human platelets (4 · 10 8
ÆmL)1) were pre-treated or not for 10 min with 10 m M EGTA or 1 m M of RGDS peptide Platelets were then stimulated with 1 UÆmL)1thrombin or
150 ngÆmL)1TPO Platelets were lysed by the addition of Triton X-100 lysis buffer at 30, 60, 120 or 240 s Triton X-100 soluble 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.
Trang 3Thombin, 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 to the insoluble fraction and secondary
responses, aggregation mediated by the integrin GPIIb-IIIa
was inhibited by EGTA and the activation mediated by
thromboxane A2and ADP were blocked by indomethacin
and apyrase, respectively Thrombin, convulxin and TPO
stimulated a concentration-dependent increase in RAS
activation (Fig 2A, C and E) Thrombin (1 UÆmL)1)
induced maximal RAS activation at 30 s which was
sustained for 240 s Convulxin (10 lgÆmL)1) induced
max-imal RAS activation at 10 s which was also sustained for
240 s TPO (150 ngÆmL)1) stimulated a gradual increase in
RAS activation which was detectable between 60 and 120 s
and maximal at 240 s (Fig 2B, D and F) These results
indicate that thrombin, convulxin and TPO induce RAS
activation, but with different time-courses
Convulxin and TPO induce GRB2 recruitment
to phosphorylated adapters
GDP–GTP exchange in RAS is regulated by the exchange
factor SOS, which is constitutively associated with the
adapter GRB2 A constitutive association between GRB2 and SOS was observed in platelets (data not shown) Tyrosine phosphorylated proteins associated with GRB2 were detected by coimmunoprecipitation and Western blotting using an anti-phosphotyrosine Ig As described previously, in convulxin-stimulated platelets, GRB2 binds
to 36, 50, 70 and 150 kDa phosphorylated-proteins ([10] and Fig 3A) In TPO-stimulated platelets only one
Thr.
WB: RAS
25 16 kDa
0 30 60 120
E
F
25 16 kDa
240 s.
10
Cvx
0 10 30 60 120 s.
TPO
0 10 30 60 120 240 s.
Thr U/ml
0 0.01 0.1 1
Cvx µ g/ml
0 0.1 1 10
TPO ng/ml
0 25 50 100 150
Pull-down: RAF-RBD; WB: RAS
Pull-down: RAF-RBD; WB: RAS
WB: RAS
25 16 kDa
25 16 kDa
Fig 2 Thrombin, convulxin and TPO induce RAS activation in a
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 for 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 by the
addition of RAS lysis buffer Cell extracts were precipitated using GST
fusion protein containing the RAS-binding domain of 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 presented are
represen-tative of three experiments.
Thr.
A
IP:GRB2, WB:P-Y
Cvx _
62
IP:GRB2, WB:GRB2
25 kDa
25 kDa
25 kDa
25 kDa
62 kDa
62
25 kDa 32
Cvx _
IP:SHC, WB:P-Y
IP:SHC, WB:GRB2
IP:GRB2, WB:SYK
IP:GRB2, WB:LAT
IP:GRB2, WB:GRB2
IP:SHC, WB:P-Y
IP:SHC, WB:GRB2
TPO
0 10 30 60 120 240 s.
175 83 62 47 32 kDa
175 83 62 47 32 kDa
Cvx
0 10 30 60 120s.
IP:GRB2, WB:P-Y
IP:GRB2, WB:GRB2
C B
E D
Fig 3 Convulxin and TPO but not thrombin induce the recruitment of GRB2 on phosphorylated adapters (A, B and C) Washed human platelets were stimulated with 1 UÆmL)1 of thrombin for 120 s,
10 lgÆmL)1convulxin for 120 s and 150 ngÆmL)1of TPO for 240 s Platelets were then lysed by addition of Nonidet P-40 lysis buffer (A) Cell extracts were immunoprecipitated using polyclonal GRB2 Ig Precipitated proteins were resolved by 10% SDS/PAGE and analysed
by Western blotting using an anti-phosphotyrosine Ig (top) The filter was stripped and reprobed using a monoclonal anti-GRB2 Ig (bot-tom) (B) Cell extracts were immunoprecipitated using an anti-SHC Ig Precipitated proteins were resolved by 10% SDS/PAGE and analysed
by Western blotting using an anti-phosphotyrosine Ig (top) or a monoclonal anti-GRB2 Ig (bottom) (C) Cell extracts were immuno-precipitated using polyclonal GRB2 Ig Precipitated proteins were resolved by 10% SDS/PAGE and analysed by Western blotting using
an anti-SYK Ig (top) or anti-LAT Ig (middle) The lower part of the filter was stripped and reprobed using a monoclonal anti-GRB2 Ig (bottom) (D) Human platelets were stimulated from 10 to 240 s with
150 ngÆmL)1of TPO and were then lysed Cell extracts were immuno-precipitated using an anti-SHC Ig Precipitated proteins were resolved
by 10% SDS/PAGE and analysed by Western blotting using an anti-phosphotyrosine Ig (top) or a monoclonal anti-GRB2 Ig (bottom) (E) Human platelets were stimulated from 10 to 120 s with 10 lgÆmL)1
of convulxin and were then lysed Cell extracts were immuno-precipitated using polyclonal anti-GRB2 Ig Precipitated proteins were resolved by 10% SDS/PAGE and analysed by Western blotting using
an anti-phosphotyrosine Ig (top) The filter was stripped and reprobed using a monoclonal anti-GRB2 Ig (bottom) Results presented are representative of three experiments.
Trang 4phosphorylated protein was detected at 50 kDa No change
in the pattern of phosphorylated proteins associated with
GRB2 was observed in platelets stimulated by thrombin
Convulxin and TPO stimulated tyrosine phosphorylation of
the 50 kDa adapter SHC, leading to formation of a
complex with GRB2 (Fig 3B) In convulxin-stimulated
platelets, GRB2 also coimmunoprecipitated with the adapter
protein LAT and the tyrosine kinase SYK, suggesting that
these phosphorylated proteins could also recruit GRB2
after stimulation (Fig 3C) Time-course studies indicated
that SHC phosphorylation and GRB2 association in
response to TPO occurred gradually 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 kinases 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 previously shown weak
phos-phorylation of ERK1 in response to thrombin and collagen
[7], only ERK2 phosphorylation was detected using the
antiphospho ERK1-2 In contrast, TPO was unable to
induce ERK1 or 2 activation (Fig 4C)
Treatment of platelets with the protein kinase C (PKC)
inhibitor Ro 31 8220 abolished phosphorylation of the
major PKC substrate pleckstrin and blocked activation of
ERK in response to thrombin and convulxin (Fig 5 A and
B) This is consistent with previous reports demonstrating
that ERK regulation is mediated downstream of PKC [7] In
contrast, activation of RAS by thrombin and convulxin was
not affected by treatment with Ro 31 8220 (Fig 5C)
suggesting that activation of RAS is not sufficient on its
own to activate ERK in platelets This is consistent with the
observation that TPO was unable to induce pleckstrin
phosphorylation or activation of ERK despite conversion of RAS to its GTP-bound form (Fig 5A and C and Fig 4C)
RAS but not ERK activation induced by thrombin
is dependent on SRC kinases SRC family kinases are necessary for the initial signalling events induced by GPVI, notably for phosphorylation of the GPVI-associated receptor FcR c-chain [11,12] As expected, the SRC kinase family inhibitor PP1 inhibited convulxin-induced ERK and RAS activation (Fig 6A,B) Interestingly, although activation of ERK in response to thrombin was not affected by PP1, activation of RAS was strongly reduced These results suggest that RAS but not ERK activation induced by thrombin is regulated by SRC
Thr.
WB: P-ERK
Ro 318220
_
Ro 318220
_ Reprobe: ERK2
WB: P-ERK
Reprobe: ERK2
Thr.
_
_
_
Cvx Cvx +Ro
TPO
Cvx
Thr _ + _ +
_ + _ +
47 kDa
Ro 318220
_
Thr _ + _ +
25 kDa
Ro 318220
_
Ro 318220
_
WB: RAS
47 kDa
WB: RAS Pull-down: RAF-RBD
WB: RAS Pull-down: RAF-RBD
Pull-down: RAF-RBD
47 kDa
47 kDa
47 kDa
47 kDa
47 kDa
25 kDa
25 kDa
25 kDa
25 kDa
25 kDa
Fig 5 PKC inhibitor Ro-31 8220 inhibited ERK but not RAS acti-vation induced by thrombin, convulxin and TPO (A, B and C) Washed platelets or [32P]orthophosphate-labelled platelets were pretreated for
10 min with 10 l M Ro 31 8220 and stimulated for 2 min with
1 UÆmL)1 thrombin, for 2 min with 10 lgÆmL)1 convulxin or for
5 min with 150 ngÆmL)1TPO (A) Stimulated labelled-platelets were lysed in Laemmli sample buffer and were resolved by 12% SDS/ PAGE Phosphorylated pleckstrin was detected by autoradiography (B) Platelets were lysed by the addition of denaturating 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 stripped and reprobed using an anti-ERK2 Ig (bottom) (C) Platelets were lysed by the addition 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 of whole cell lysate were resolved by 12.5% SDS/PAGE and analysed by Western blotting using anti-RAS
Ig (bottom) Results presented are representative of three experi-ments.
Thr.
WB: P-ERK
47 kDa
0 30 60 120 s.
Reprobe: ERK2
47 kDa
2 Cvx
Fig 4 Thrombin and convulxin but not TPO induce ERK activation.
(A, B and C) Washed human platelets were stimulated for the times
indicated with 1 UÆmL)1 thrombin, 10 lgÆmL)1 convulxin or
150 ngÆmL)1TPO Platelets were then lysed by addition of
denatu-rating 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 stripped and reprobed using
an anti-ERK2 Ig (bottom) Results presented are representative of
three experiments.
Trang 5family kinases and suggest that activation of RAS is
dispensable for efficient activation of ERK
D I S C U S S I O N
Activation of RAS was evaluated in platelets through the
ability of the activated RAS–GTP to bind a GST fusion
protein containing the RAS-binding domain of RAF
Different platelet agonists including thrombin, the snake
venom convulxin and TPO were able to activate RAS It has
been shown that H-RAS is expressed in platelets, but
expression of the isoform ki- and N-RAS was not excluded
[5] Activated RAS was detected with an anti-(pan RAS) Ig,
which does not distinguish the various isoforms of RAS In
platelets, SOS, the exchange factor of RAS, was found in complex with the adapter GRB2 In response to convulxin, GRB2 associates with a number of phosphorylated pro-teins Three of these phosphorylated proteins, the adapters LAT and SHC, and the tyrosine kinase SYK were identified These proteins belong to the signalling complex activated by phosphorylation in response to the cross-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 shown 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 to phosphorylated adapters, suggest-ing a possible regulation 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 of RAS, suggesting a causal relationship In megakaryocytic cell lines, it has been shown that GRB2 recruitment by phosphorylated SHC following c-Mpl receptor activation
by TPO contributes to activation of the RAS–ERK signalling pathway [16]
The SRC kinase family inhibitor PP1 inhibited activa-tion of RAS in response to thrombin Activaactiva-tion of RAS
by G coupled-receptors in other cell types is also mediated through the SRC kinases [17–19] PP1 also inhibited activation of RAS stimulated by convulxin, consistent with the role of these kinases in mediation 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 by sequential activation of RAS– RAF–MEK and ERK In platelets, phosphorylation of ERK2 in response to thrombin and GPVI agonists is dependent on PKC ([7] and this study) In contrast, we found that a PKC inhibitor did not affect RAS activity in response to thrombin and convulxin This is consistent with the observation that TPO, which is unable to induce activation of PKC, does not stimulate activation of ERK in platelets This result indicates that activation of RAS is not sufficient on its own to lead to ERK activation The differential regulation of RAS and ERK was also shown by the different time-courses of activation between these two proteins in response to thrombin and convulxin
In a number of other cells, PKC is also able to regulate ERK activity In most of these cases, PKC activates RAF, which in turn activates the MEK–ERK cascade, but activation of ERK without involvement of RAF has also been shown [20,21] In platelets, the mechanism of ERK activation by PKC has not been elucidated However, activation of ERK by thrombin, GPVI agonists and phorbol ester is abolished by inhibitors of MEK ([22] and data not shown) suggesting that this activation occurs at the level of, or upstream of, MEK In addition, it has been shown that phorbol ester, which induces efficient activation
of ERK, is not able to induce RAF activation in platelets [6] Taken together these results suggest that regulation of ERK by PKC is not mediated through RAF but more likely through activation of MEK
Fig 6 SRC inhibitor PP1 inhibited RAS but not ERK activation
induced by thrombin (A and B) Human platelets were pretreated for
10 min with 10 l M PP1 and stimulated for 2 min with 1 UÆmL)1
thrombin, for 2 min with 10 lgÆmL)1convulxin or for 5 min with
150 ngÆmL)1TPO (A) Platelets were lysed by 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 stripped and reprobed using
an anti-ERK2 Ig (bottom) (B) Platelets were lysed by the addition 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 of whole cell lysate were
resolved by 12.5% SDS/PAGE and analysed by Western blotting
using anti-RAS Ig (bottom) Results presented are representative of
three experiments.
Trang 6Nevertheless, RAS could participate in the regulation of
ERK by 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 ability of TPO to
activate the early events of the RAS signalling pathway
However, a recent study reported that inhibitors of
PtdIns3K abolished potentiation of ERK by TPO in
response to thrombin, demonstrating that potentiation is
mediated though the PtdIns3K pathway The authors
proposed a model in which activation of PtdIns3K by
TPO potentiates activation of MEK induced by thrombin,
which in turn potentiates activation of ERK [23] Whether
this set of events involves activation of RAS is not clear
TPO is also able to potentiate aggregation and secretion
induced by thrombin Interestingly, although inhibitors of
PtdIns3K abolished this potentiation, the inhibitor of MEK
had no or only a weak effect, suggesting that the MEK–
ERK cascade is not the main downstream event induced by
PtdIns3K to potentiate these functional responses [23]
All of the known components of the RAS–ERK
signalling pathway (RAS, RAF, MEK and ERK) are
expressed in platelets Furthermore, these proteins can be
activated independently, suggesting that each link is
func-tional [5–7] In addition, in megakaryocytic cell lines and in
primary megakaryocytes, the precursor of platelets, the
RAS–ERK signalling pathway is activated by TPO and is
essential for differentiation [16,25] In the last few years, a
number of other proteins able to regulate the RAS–ERK
pathway have been described This includes scaffolding
proteins, which promote interaction between the links of the
pathway For instance, MP1 can bind MEK and ERK1
supporting ERK1 activation [26], and kinase suppressor of
RAS can bind RAF, MEK and ERK favouring activation
of MEK and ERK1-2 through RAS [27] On the other
hand, the RAS–ERK signalling pathway can be
down-regulated by proteins able to bind to its components For
example, the RAF kinase inhibitor protein can bind RAF
and MEK and thereby prevent their interaction [28,29] A
reduction of the expression of the scaffolding proteins or an
over-expression of proteins responsible for down-regulation
could be an explanation for the inefficiency of RAS to
activate ERK in platelets
RAS–RAF interaction and subsequent 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 target
ERK Platelets appear to be a model with which to study
down-regulation of the RAS–ERK signalling pathway 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
A C K N O W L E D G E M E N T S
This work was supported by the British Heart Foundation (BHF).
S P W is a BHF Senior Research Fellow.
R E F E R E N C E S
1 Buday, L & Downward, J (1993) Epidermal growth factor regu-lates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor Cell 73, 611–620.
2 Egan, S.E., Giddings, B.W., Brooks, M.W., Buday, L., Sizeland, A.M & Weinberg, R.A (1993) Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal trans-duction and transformation Nature 363, 45–51.
3 Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T & Bowtell, D (1993) The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1 Nature
363, 83–85.
4 McCormick, F & Wittinghofer, A (1996) Interactions between Ras proteins and their effectors Curr Opin Biotechnol 7, 449–456.
5 Shock, D.D., He, K., Wencel-Drake, J.D & Parise, L.V (1997) Ras activation in platelets after stimulation of the thrombin receptor, thromboxane A2 receptor or protein kinase C Biochem.
J 321, 525–530.
6 Ezumi, Y., Uchiyama, T & Takayama, H (1998) Thrombo-poietin potentiates the protein-kinase-C-mediated activation of mitogen-activated protein kinase/ERK kinases and extracellular signal-regulated kinases in human platelets Eur J Biochem 258, 976–985.
7 Borsch-Haubold, A.G., Kramer, R.M & Watson, S.P (1995) Cytosolic phospholipase A2 is phosphorylated in collagen- and thrombin- stimulated human platelets independent of protein kinase C and mitogen-activated protein kinase J Biol Chem 270, 25885–25892.
8 Pasquet, J.M., Quek, L., Stevens, C., Bobe, R., Huber, M., Duronio, V., Krystal, G & Watson, S.P (2000) Phosphatidy-linositol 3,4,5-trisphosphate regulates Ca2+ entry via Btk in platelets and megakaryocytes without increasing phospholipase C activity EMBO J 19, 2793–2802.
9 Gibbins, J., Asselin, J., Farndale, R., Barnes, M., Law, C.-L & Watson, S.P (1996) Tyrosine phosphorylation of the Fc receptor c-chain in collagen-stimulated platelets J Biol Chem 271, 18095– 18099.
10 Asazuma, N., Wilde, J.I., Berlanga, O., Leduc, M., Leo, A., Schweighoffer, E., Tybulewicz, V., Bon, C., Liu, S.K., McGlade, C.J., Schraven, B & Watson, S.P (2000) Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin.
J Biol Chem 275, 33427–33434.
11 Briddon, S.J & Watson, S.P (1998) Evidence for the involvement
of p59 fyn and p53/56 lyn in signalling via the collagen receptor in human platelets Biochem J 337, 203–209.
12 Quek, L.S., Pasquet, J.M., Hers, I., Cornall, R., Knight, G., Barnes, M., Hibbs, M.L., Dunn, A.R., Lowell, C.A & Watson, S.P (2000) Fyn and lyn phosphorylate the Fc receptor gamma chain downstream of glycoprotein VI in murine platelets, and lyn regulates a novel feedback pathway Blood 96, 4246–4253.
13 Watson, S.P & Gibbins, J (1998) Collagen receptor signalling in platelets: extending the role of the ITAM Immunol Today 19, 260–265.
14 Ravichandran, K.S., Lee, K.K., Songyang, Z., Cantley, L.C., Burn, P & Burakoff, S.J (1993) Interaction of Shc with the zeta chain of the T cell receptor upon T cell activation Science 262, 902–905.
15 Finco, T.S., Kadlecek, T., Zhang, W., Samelson, L.E & Weiss, A (1998) LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway Immunity 9, 617–626.
16 Rojnuckarin, P., Drachman, J.G & Kaushansky, K (1999) Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role
in endomitosis Blood 94, 1273–1282.
Trang 717 Sadoshima, J & Izumo, S (1996) The heterotrimeric G q
protein-coupled angiotensin II receptor activates p21 ras via the tyrosine
kinase-Shc-Grb2-Sos pathway in cardiac myocytes EMBO J 15,
775–787.
18 Luttrell, L.M., Hawes, B.E., van Biesen, T., Luttrell, D.K.,
Lansing, T.J & Lefkowitz, R.J (1996) Role of c-Src tyrosine
kinase in G protein-coupled receptor- and Gbetagamma
subunit-mediated activation of mitogen-activated protein kinases J Biol.
Chem 271, 19443–19450.
19 Luttrell, L.M., Della Rocca, G.J., van Biesen, T., Luttrell, D.K &
Lefkowitz, R.J (1997) Gbetagamma subunits mediate
Src-dependent phosphorylation of the epidermal growth factor
receptor A scaffold for G protein-coupled receptor-mediated Ras
activation J Biol Chem 272, 4637–4644.
20 Grammer, T.C & Blenis, J (1997) Evidence for
MEK-independent pathways regulating the prolonged activation of the
ERK-MAP kinases Oncogene 14, 1635–1642.
21 Bapat, S., Verkleij, A & Post, J.A (2001) Peroxynitrite activates
mitogen-activated protein kinase (MAPK) via a
MEK-independent pathway: a role for protein kinase C FEBS Lett 499,
21–26.
22 Borsch-Haubold, A.G., Kramer, R.M & Watson, S.P (1996)
Inhibition of mitogen-activated protein kinase does not
impair primary activation of human platelets Biochem J 318,
207–212.
23 Kojima, H., Shinagawa, A., Shimizu, S., Kanada, H., Hibi,
M., Hirano, T & Nagasawa, T (2001) Role of
phosphatidyli-nositol-3 kinase and its association with Gab1 in
thrombopoietin-mediated up-regulation of platelet function Exp Hematol 29,
616–622.
24 Bobe, R., Wilde, J.I., Maschberger, P., Venkateswarlu, K., Cullen,
P.J., Siess, W & Watson, S.P (2001) Phosphatidylinositol
3-kinase-dependent translocation of phospholipase Cgamma2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation Blood 97, 678–684.
25 Rouyez, M.C., Boucheron, C., Gisselbrecht, S., Dusanter-Fourt,
I & Porteu, F (1997) Control of thrombopoietin-induced mega-karyocytic differentiation by the mitogen-activated protein kinase pathway Mol Cell Biol 17, 4991–5000.
26 Schaeffer, H.J., Catling, A.D., Eblen, S.T., Collier, L.S., Krauss,
A & Weber, M.J (1998) MP1: a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade Science
281, 1668–1671.
27 Therrien, M., Michaud, N.R., Rubin, G.M & Morrison, D.K (1996) KSR modulates signal propagation within the MAPK cascade Genes Dev 10, 2684–2695.
28 Yeung, K., Seitz, T., Li, S., Janosch, P., McFerran, B., Kaiser, C., Fee, F., Katsanakis, K.D., Rose, D.W., Mischak, H., Sedivy, J.M & Kolch, W (1999) Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP Nature 401, 173–177.
29 Yeung, K., Janosch, P., McFerran, B., Rose, D.W., Mischak, H., Sedivy, J.M & Kolch, W (2000) Mechanism of suppression
of the Raf/MEK/extracellular signal-regulated kinase path-way by the raf kinase inhibitor protein Mol Cell Biol 20, 3079–3085.
30 Joneson, T., White, M.A., Wigler, M.H & Bar-Sagi, D (1996) Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS Science 271, 810–812.
31 Rodriguez-Viciana, P., Warne, P.H., Khwaja, A., Marte, B.M., Pappin, D., Das, P., Waterfield, M.D., Ridley, A & Downward, J (1997) Role of phosphoinositide 3-OH kinase in cell transforma-tion and control of the actin cytoskeleton by Ras Cell 89, 457–467.