Báo cáo Y học: Regulation of RAS in human platelets Evidence that activation of RAS is not sufficient to lead to ERK1-2 phosphorylation pot

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. Keywords: ERK; glycoprotein VI; platelet; protein kinase C RAS; signalling; thrombin; t hrombopoietin. RAS is a ubiquitously expressed G TPase protein, w hich is activated following conversion from a GDP to GTP-bound state. GDP –GTP exchange is stimulated by SOS, which is constitutively associated throu gh its proline rich domain to the S H3 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. Loc alization 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, GAP p120 , 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 M EK, 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 underg o activation. It has been shown that the G protein-c oupled receptor agonists thromboxane A 2 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 M sodium citrate/ 110 m M glucose/80 m 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,gluthationeS-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 1 9 October 200 1, revised 1 November 2001, accepted 21 January 2 002) Eur. J. Biochem. 269, 1511–1517 (2002) Ó FEBS 2002 plasma was obtained by centrifugation at 200 g for 20 min. Platelets were isolated from platelet-rich plasma by ce ntrif- ugation at 1000 g for 10 min, in the presence of prostacyclin (0.1 lgÆmL )1 ). The pellet w as resuspended i n 25 mL of a modified T yrode’s/Hepes buffer ( 134 m M NaCl, 0 .34 m M Na 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 Platelets (4 · 10 8 cellsÆmL )1 )treatedfor10minwith 2UÆmL )1 apyrase, 10 l M indomethacin and 1 m M EGTA were lysed with an equal volume of ice-cold Nonidet P-40 buffer [ 20 m M Tris, 300 m M NaCl, 2 m M EDTA, 2 % ( v/v) NP40, 1 m M phenylmethanesulfonyl fluoride, 2 m M Na 3 VO 4, 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 °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 were lysed with an equal volume of ice-cold Nonidet P-40 buffer containing additional 1% N-octyl glucoside and 5m M MgCl 2 . Lysed cells and debris were removed by centrifugation. Ce ll lysates were precleared for 1 h at 4 °C with glutathione–agarose. Platelet lysates were incubated 3hat 4°Cwith5lg G ST–RAF–RBD immobilized on agarose. The pellet was washed once in lysis buffer and three times in NaCl/Tris/Tween. Immunoblotting For ERK ph osphorylation, platelets w ere lysed in 10 · de- naturating buffer (10% SDS, 100 m M NaCl, 50 m M Tris, 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 ch emi- luminescence detection (ECL) system. Cytoplasmic and cytoskeleton localization Platelets were p repared and stimulated as described a bove and then lysed with an equal volume of ice-cold Triton X-100 lysis buffer (100 m M 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 8 plateletsÆmL )1 in Tyrode’s/Hepes plus indomethacin and left 15 min before the e xperiment as described above. Reactions were stopped using Laemmli buffer. Proteins were resolved on SDS/ PAGE and visualized by autoradiography. RESULTS 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 i n response t o thrombin induced translocation of RAS from the cytoplasmic f raction to the cytoskeleton fraction (Fig. 1A). In the presence of EGTA or RGDS peptide, both of which inhibit G PIIb-IIIa-dependent aggre- gation, relocalization of RAS was not observed ( Fig. 1B and C). A similar set of results was obtained in r esponse to aggregation induced by the GPVI agonist, c onvulxin (data not shown). TPO does not induce aggregation o r 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 c ytoplasmic to t he cytoskeleton fraction. (A,B,CandD)Washedhumanplatelets(4· 10 8 ÆmL )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 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 d etectable between 60 and 120 s and maximal at 2 40 s (Fig. 2B, D and F). These results indicate th at thrombin, convulxin a nd 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 µ 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 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 convulxin for 120 s and 150 ng ÆmL )1 of TPO for 240 s. Platelets were t hen lysed by addition of Nonidet P-40 lysis bu ffer. (A) Cell extracts were immunoprecipitated using polyclonal GRB2 I g. Precipitated prot eins were resolved b y 10% SDS/PAGE and analysed by Western blo tting using an anti-phosphotyrosine Ig (top). The filter was s tripped and re probed using a monoclonal anti-GRB2 Ig (bot- tom). (B) Cell extracts were immunoprecipitated using a n anti-SHC I g. Precipitated prot eins were resolved b y 10% SDS/PAGE and analysed by Western blotting usin g an a nti-phosphotyrosine I g (top ) or a monoclonal anti-GRB2 Ig (bottom). (C) Cell ext racts w ere immuno- precipitated using polyclonal G RB2 I g. Precipitated proteins were resolved by 10% SDS/P AGE and a nalysed by W estern blotting using an anti-SYK Ig (top) or anti-L AT Ig (m iddle). The lower part of the filter was stripped and r eprobed using a mon oclonal anti-GRB2 Ig (bottom). (D) Human platelets were stimulated from 10 to 240 s with 150 ngÆmL )1 of 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 b lotting 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 c onvulxin and were then lysed. Cell extracts were immuno- precipitated using polyclonal anti-GRB2 Ig. Precipitated proteins were resolved by 10% SDS/P AGE and a nalysed by W estern blotting using an anti-phosphotyrosine Ig (top). Th e filter was stripped and reprobed using a monoclonal anti-GRB2 Ig (bottom). Results presented are 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 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 th rombin 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 b y thrombin and convulxin was not affected by treatment with Ro 31 8220 (Fig. 5C) suggesting t hat activation o f R AS is not sufficient o n i ts own to activate ERK in platelets. This is consistent with the observation that TPO was unable to induce pleckstrin phosphorylation or a ctivation of ERK despite conversion of RAS to its GTP-bound form (Fig. 5A a nd C a nd 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 G PVI-associated rece ptor FcR c-chain [11,12]. A s 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, a ctivation of RAS was strongly reduced. These results suggest that RAS but not ERK activation induced by thrombin is regulated by SRC 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 o r [ 32 P]orthophosphate-labelled p latelets were pretreated for 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 )1 convulxin or 150 n g ÆmL )1 TPO. Platelets were then lysed by addition of denatu- rating lysis buffer. P roteins of whole cell lys ate were resolved by 10% SDS/PAGE and a nalysed b y W est ern blo tting u sing a n a nti-(pho spho- specific ERK1-2) Ig (top). The filter w as stripped and reprobed using an anti-ERK2 Ig (bottom). Results presented are representative of three experiments. 1514 D. Tulasne et al. (Eur. J. Biochem. 269) Ó FEBS 2002 family kinases and suggest that activation of R AS is dispensable for efficient activation of ERK. DISCUSSION Activation of RAS was evaluated in platelets through the ability of t he activated RAS–GTP to b ind a GST f usion 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 w as 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 w ith the adapter GRB2. In response to convulxin, GRB2 associates with a number of phosphorylated pro- teins. Three of these phosphorylated proteins, t he 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 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 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 sufficientonitsowntoleadtoERKactivation.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, P KC is also able to regulate ERK activity. In most of these cases, PKC activates RAF, whichinturnactivatestheMEK–ERKcascade,but activation of ERK without involvement of RAF has also been shown [20,21]. In platelets, t he 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 p latelets [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 a nd B ) H um an pla telet s w ere p retreated for 10 min with 1 0 l M 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 proposed a model in which activation of PtdIns3K by TPO potentiates activation of MEK induced by thrombin, whichinturnpotentiatesactivationofERK[23].Whether this set of events involves activation of RAS is not clear. TPO i s a lso able to potentiate aggregation a nd 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 T PO 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. F or instance, MP1 can bind MEK and ERK1 supporting ERK1 activation [26], and kinase suppressor o f 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 M EK 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 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. 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Regulation of RAS in human platelets Evidence that activation of RAS is not sufficient to lead to ERK1-2 phosphorylation David Tulasne 1 ,. 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