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Oncogene (1999) 18, 2047 ± 2054 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc Eect of SB 203580 on the activity of c-Raf in vitro and in vivo Clare A Hall-Jackson*,1, Michel Goedert2, Philip Hedge3 and Philip Cohen1 1 MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, Scotland; 2MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England; 3Zeneca Pharmaceuticals, Alderley Edge, Maccles®eld, Cheshire SK10 4TG, England The inhibition of SAPK2a/p38 (a mitogen activated protein (MAP) kinase family member) by SB 203580 depends on the presence of threonine at residue 106. Nearly all other protein kinases are insensitive to this drug because a more bulky residue occupies this site (Eyers et al., 1998). Raf is one of the few protein kinases that possesses threonine at this position, and we show that SB 203580 inhibits c-Raf with an IC50 of 2 mM in vitro. However, SB 203580 does not suppress either growth factor or phorbol ester-induced activation of the classical MAP kinase cascade in mammalian cells. One of the reasons for this is that SB 203580 also triggers a remarkable activation of c-Raf in vivo (when measured in the absence of the drug). The SB 203580-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras, is not prevented by inhibitors of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase, and is not triggered by the binding of this drug to SAPK2a/p38. The paradoxical activation of cRaf by SB 203580 (and by another structurally unrelated c-Raf inhibitor) suggests that inhibitors of the kinase activity of c-Raf may not be eective as anti-cancer drugs. Keywords: c-Raf; MAP kinase; SB203580; EGF; SAPK2; p38 Introduction Protein kinases form one of the largest protein families encoded in the human genome, and many of these enzymes have become potential targets for drug therapy because abnormal protein phosphorylation is a cause or a consequence of many diseases. Several relatively speci®c inhibitors of particular protein kinases have been developed that have potential for the treatment of cancer (Law and Lydon, 1996), diabetes (Ishii et al., 1996), and hypertension (Uehata et al., 1997). In addition, a class of pyridinyl imidazoles has been identi®ed that suppress the synthesis (and some of the actions) of proin¯ammatory cytokines (Lee et al., 1994) and show promise for the treatment of rheumatoid arthritis and other chronic in¯ammatory conditions (Badger et al., 1996). The pyridinyl imidazole SB 203580 is an inhibitor of two stress-activated protein kinases (SAPKs) termed SAPK2a (or p38an) and SAPK2b (or p38b2) (Cuenda *Correspondence: CA Hall-Jackson Received 2 July 1998; revised 5 November 1998; accepted 11 December 1998 et al., 1995; Goedert et al., 1997). These enzymes, which display 75% amino acid sequence identity, are members of the mitogen-activated protein (MAP) kinase family, but other members of this gene family are insensitive to SB 203580. These include SAPK3 (p38g) and SAPK4 (p38d) whose amino acid sequences are 60% identical to SAPK2a/p38 or SAPK2b/p38b2 (Goedert et al., 1997). SB 203580 also fails to inhibit many other protein kinases tested (Cuenda et al., 1995) and, for this reason, it has been used extensively to identify substrates and physiological roles of SAPK2/ p38 (reviewed in Cohen, 1997). SB 203580 binds competitively with ATP (Young et al., 1997) and the three-dimensional structure of SAPK2a/p38a in a complex with closely related pyridinyl imidazoles has established that these drugs are inserted into the ATP-binding pocket of SAPK2a/ p38a (Tong et al., 1997; Wilson et al., 1997). However, the 4-¯uorophenyl ring of the drug does not make contact with residues in the ATP-binding pocket that interact with ATP. One residue that makes contact with the 4-¯uorophenyl moiety is Thr 106 and recent studies have established that this residue plays a critical role in determining sensitivity to SB 203580. Thus its mutation to Met or Gln, which are present at the equivalent position in other MAP kinase family members, or to other residues with bulky side chains, makes SAPK2a/p38a and SAPK2b/p38b2 insensitive to SB 203580 (Wilson et al., 1997; Eyers et al., 1998; Gum et al., 1998). Conversely, the mutation of this residue to Thr in other MAP kinase family members (SAPK1/JNK, SAPK3/p38g and SAPK4/p38d) makes them sensitive to SB 203580 (Eyers et al., 1998; Gum et al., 1998). Sensitivity to SB 203580 is greatly enhanced when the side chain at residue 106 is smaller than Thr (i.e. Ser, Ala or Gly). Examination of the sequences of protein kinases in the data bases reveals that a bulky residue is almost always found at the position equivalent to Thr 106, explaining why nearly all protein kinases are insensitive to SB 203580. However, a small number of protein kinases do have Thr at this position, and we have shown that two of these enzymes, the Type-II TGFb receptor and the protein tyrosine kinase lck, are inhibited by SB 203580 (Eyers et al., 1998). In our standard assay, conducted at 0.1 mM ATP, the IC50 values for the Type-II TGFb receptor and lck are 40 and 20 mM, respectively, 400 ± 800-fold higher than the IC50 for inhibition of SAPK2a/p38a and 40 to 80-fold higher than the IC50 for inhibition of SAPK2b/p38b2. Nevertheless, the Type-II TGFb receptor becomes insensitive to SB 203580 when the Thr is changed to Met, and is inhibited 10-fold more potently when this residue is changed to Ala (Eyers et al., 1998). Thus the sensitivity of the Type-II TGFb receptor to SB 203580 Effect of SB203580 on Raf CA Hall-Jackson et al 2048 also depends on the presence of Thr at the position equivalent to Thr 106 of SAPK2a/p38. Two mammalian protein kinases (the Type-I TGFb receptor and an Activin receptor) have Ser and not Thr at this position. The Type-I TGFb receptor is inhibited by SB 203580 (IC50=20 mM) and sensitivity is abolished when this residue is mutated to Met (Eyers et al., 1998). No protein kinases in the public data bases have Ala or Gly at this position. SB 203580 does not aect the activation of the classical MAP kinase cascade by a variety of agonists (Cuenda et al., 1995; Gould et al., 1995; Hazzalin et al., 1997). However, the protein kinase that is thought to lie at the head of this cascade, the proto-oncogene cRaf, contains threonine at the position equivalent to Thr 106 of SAPK2a/p38a. Here we show that c-Raf is inhibited by SB 203580 in vitro with an IC50 of 2 mM, a concentration that is 40-fold higher than the IC50 for SAPK2a/p38a, but only fourfold higher than the IC50 for SAPK2b/p38b2. The failure of SB 203580 to suppress activation of the MAP kinase cascade in vivo may be related to a remarkable activation of c-Raf that occurs when mammalian cells are incubated with this drug. Results Eect of SB 203580 on activated c-Raf in vitro The sensitivity of protein kinases to SB 203580 depends on the presence of threonine, or a smaller residue, at the position equivalent to Thr 106 of SAPK2a/p38a (see Introduction). Since c-Raf is one of a small number of protein kinases that possess threonine at this position, we initially examined the eect of SB 203580 on human c-Raf that had been activated in insect Sf9 cells by cotransfection of its DNA with DNA encoding v-Ras and the protein tyrosine kinase Lck. c-Raf in Sf9 cell extracts is inhibited by SB 203580 with an IC50 value of 2 mM (Figure 1). This is 40-fold higher than the IC50 for SAPK2a/p38a, but only four-fold higher than the IC50 for SAPK2b/p38b2 (Eyers et al., 1998). However, the IC50 values became 10-fold higher if the assays were carried out using c-Raf immunoprecipitated from Sf9 cells (Figure 1), EGF-stimulated mouse Swiss 3T3 cells (Figure 1) or EGF-stimulated 293 cells (data not shown). Similar results were obtained if the Swiss 3T3 cells or the 293 cells were stimulated with PMA (data not shown). Possible reasons for this dierence in sensitivity to SB 203580 are considered further under Discussion. The human B-Raf isoform was also expressed in Sf9 cells and was found to be inhibited with an IC50 of 7 mM when assayed in the Sf9 cell lysates without immunoprecipitation (Results not shown). SK&F 105809 (10 mM), a compound that is closely related in structure to SB 203580 but does not inhibit SAPK2a/p38a, also did not inhibit c-Raf or B-Raf activity (data not shown). c-Raf and B-Raf are assayed in a coupled assay containing MKK1 and MAPK2. Neither of these coupling enzymes are inhibited by SB 203580 (Cuenda et al., 1995). The drug PD 98059 binds to MKK1 preventing its phosphorylation and activation by c-Raf (Alessi et al., Figure 1 Inhibition of c-Raf activity by SB 203580 in vitro. Human c-Raf was activated in Sf9 cells by cotransfection with DNA encoding v-Ras and Lck, and activity was either measured directly in the cell lysates (*) or after immunoprecipitation (*). c-Raf was also activated by incubation of mouse Swiss 3T3 cells for 2 min with 100 ng/ml EGF and assayed after immunoprecipitation (~). The results are presented relative to control incubations in which SB 203580 was omitted (mean+s.e.m. for three separate experiments) 1995a). Therefore, SB 203580 could inhibit c-Raf activity either by binding to c-Raf or by binding to MKK1. We distinguished between these possibilities, by studying the eect of SB 203580 on the autophosphorylation of c-Raf that occurs when immunoprecipitates are incubated with MgATP. The autophosphorylation of c-Raf was prevented by SB 203580 at concentrations similar to those that prevented the activation of MKK1 (data not shown), indicating that the drug exerts its eect by interacting with c-Raf directly. Failure of SB 203580 to prevent the activation of MKK1 or MAPK2 in mammalian cells The ®nding that c-Raf is inhibited by SB 203580 at micromolar concentrations was surprising because we have reported previously that the activation of MAPK1 and MAPK2 by NGF in rat PC12 cells (Cuenda et al., 1995), by EGF or IGF-1 in human KB cells (Cuenda et al., 1995; Gould et al., 1995), or by EGF or phorbol esters in mouse C3H 10T1/2 cells (Hazzalin et al., 1997), is not inhibited by SB 203580. In the present study we con®rmed this result for EGFstimulated Swiss 3T3 cells (Figure 2) and IGF-1 stimulated rat L6 myotubes (data not shown). We also showed for the ®rst time that SB 203580 has no eect on the activation of MKK1 by EGF in Swiss 3T3 cells (Figure 2) at a concentration (10 mM) where it inhibits the activity of c-Raf by 80% in vitro (Figure 1). Thus the failure of SB 203580 to inhibit the activation of MAPK2 is not explained by the activation of a MAPK phosphatase. Effect of SB203580 on Raf CA Hall-Jackson et al 2049 Figure 2 Eect of SB 203580 on the activation of MKK1 and MAPK2 in vivo. Con¯uent Swiss 3T3 cells were incubated for 18 h in DMEM containing 0.5% FCS and then for 60 min with or without 10 mM SB 203580 or 50 mM PD 98059 followed by stimulation for 5 min with 100 ng/ml EGF. The cells were lysed and MKK1 and MAPK2 activities measured after immunoprecipitation. The results are presented as a percentage of the activity obtained after stimulation for 5 min with EGF, and are presented as the mean+s.e.m. for three separate experiments. The batch of PD 98059 used in these experiments inhibits the activation of MKK1 more potently that the sample used in our earlier work, which (at 50 mM) did not suppress the EGF-induced activation of MAPK2 (Alessi et al., 1995a) Paradoxical activation of c-Raf by SB 203580 in mammalian cells In order to investigate why SB 203580 does not prevent the growth factor or PMA-induced activation of MKK1 or MAPK2, we exposed Swiss 3T3 cells to SB 203580 and then measured c-Raf activity in the absence of SB 203580 after its immunoprecipitation from the cell lysates. These experiments revealed that SB 203580 had induced a remarkable increase in c-Raf activity which was elevated about 30-fold after 60 min (Figure 3a). The activation was similar to that attained after stimulation for 2 min with EGF (Figure 3a), which is the most potent activator of c-Raf in Swiss 3T3 cells (Alessi et al., 1995a). The activation of c-Raf by EGF was transient, peaking after 2 min and returning to near basal levels after 20 min (Figure 3a), but the activation of c-Raf by SB 203580 was sustained for at least 60 min (Figure 3a). Similar results were obtained in 293 cells, where exposure to SB 203580 also increased c-Raf activity 30-fold between 6 and 60 min (results not shown). In L6 myotubes, SB 203580 increased c-Raf activity 15-fold after 60 min, similar to the activation of c-Raf induced by stimulation for 5 min with IGF-1 (results not shown). c-Raf that had been activated by incubation of Swiss 3T3 cells with 10 mM SB 203580 and then immunoprecipitated from the lysates, was just as sensitive to SB 203580 as c-Raf immunoprecipitated from the lysates of cells that had been stimulated by EGF (data not shown). SK&F 105809 (10 mM), which is closely related in structure to SB 203580 but does not inhibit SAPK2a/p38a, was unable to induce any activation c-Raf when Swiss 3T3 cells were incubated for up to 60 min (data not shown). Pretreatment of Swiss 3T3 cells for 60 min with SB 203580 (in the absence of growth factors or PMA) failed to induce any activation of MAPK2, but slightly enhanced EGF-dependent activation of c-Raf (Figure 3a) and MAPK2 (Figure 3b). Pretreatment with SB 203580 also failed to induce any activation of MAPK2 in 293 cells or L6 myotubes (results not shown). Mechanism of activation of c-Raf by SB 203580 We have reported previously that PD 98059, a drug that binds to MKK1 and thereby prevents its activation by c-Raf, also stimulates the basal activity of c-Raf several-fold as well as the rate and extent of cRaf activation by PDGF (Alessi et al., 1995a). These ®ndings suggest that the activity of c-Raf is suppressed by an activated `downstream' component of the classical MAP kinase pathway. However, SB 203580 does not induce any activation of MKK1 or MAPK2 (Figure 2). Moreover, incubation of Swiss 3T3 cells with PD 98059 (50 mM), which prevents the activation of MAPK2 by EGF (Figure 2), failed to prevent the much larger activation of c-Raf induced by SB 203580 (Figure 4). Thus activation of c-Raf by SB 203580 occurs independently of the activation of the MAP kinase cascade. The activation of c-Raf by dierent agonists can result from the stimulation of several signalling pathways, including the activation of protein kinase C (PKC) (Morrison and Cutler, 1997) or phosphatidylinositide (PtdIns) 3-kinase (Cross et al., 1994). However, c-Raf activation is unaected by Ro 318220 (Figure 4), an inhibitor of PKC and some other protein kinases (Alessi, 1997), under conditions where the PMA-induced activation of MAPK2 measured in the same cells was decreased by 85%. Similarly wortmannin, an inhibitor of PtdIns 3-kinase, does not aect the activation of c-Raf by SB 203580 under conditions Effect of SB203580 on Raf CA Hall-Jackson et al 2050 Figure 4 Eect of PD 98059, wortmannin and Ro 318220 on the activation of c-Raf by SB 203580 in Swiss 3T3 cells. Con¯uent cells were incubated in DMEM containing 0.5% FCS for 18 h and then pretereated with (+) or without (7) 50 mM PD 98059 (60 min), 100 nM wortmannin (10 min) or 0.3 mM Ro 318220 (60 min) prior to incubation for 60 min with 10 mM SB 203580 in the continued presence of these compounds. The results are presented as a percentage of the activity obtained after incubation for 60 min with SB 203580 in the absence of any other compound Figure 3 Eect of SB 203580 on the activation of c-Raf and MAPK2 in vivo. Con¯uent Swiss 3T3 cells were incubated on 18 h in DMEM containing 0.5% FCS then incubated for the times indicated with 10 mM SB 203580 (*) or 100 ng/ml EGF (*). Stimulation with EGF was also carried our after pretreatment for 60 min with 10 mM SB 203580 (~). The activities of c-Raf (a) and MAPK (b) were then measured after immunoprecipitation from the cell lysates. The results are presented as the mean+s.e.m. for three separate experiments where wortmannin suppresses IGF-1-induced activation of protein kinase B by 95%. Thus c-Raf activation induced by SB 203580 does not appear to occur via either of these pathways. The activation of c-Raf by growth factors, such as EGF, is dependent on the activation of Ras. In unstimulated Swiss 3T3 cells the % of GTP-Ras relative to GDP-Ras+GTP-Ras is 10.7+2.6%, which increases to 44+2.4% after stimulation for 2 min with EGF (Figure 5). However, after exposure to SB 203580 for 60 min, at which time c-Raf activation is maximal (Figure 2), the proportion of GTP-Ras does not increase at all (10.3+0.5%) (Figure 5). There was also no increase in GTP-Ras after exposure of the cells to SB 203580 for 2 or 10 min (data not shown). Thus the activation of c-Raf by SB 203580 does not result from an increase in the level of GTP-Ras. Prolonged mitogenic stimulation leads to the hyperphosphorylation of c-Raf, which can be visua- Figure 5 Lack of eect of SB 203580 on GTP-loading of Ras in Swiss 3T3 cells. Cells were 32p-labelled as described in Materials and methods before stimulation with 100 ng/ml EGF (2 min) or 10 mM SB 203580 (60 min). Ras was immunoprecipitated from the cell lysates and the bound guanine nucleotides eluted and separated by thin layer chromatography. The ®gure shows an autoradiograph of a representative experiment. Quantitation (+s.e.m. for three separate experiments) is presented in the text lised by a reduction in its electrophoretic mobility. However, pretreatment of Swiss 3T3 cells with SB 203580 for 6 ± 200 min does not induce any change in the electrophoretic mobility of c-Raf (results not shown). Effect of SB203580 on Raf CA Hall-Jackson et al 2051 Figure 6 Eect of SB 203580 on the activities of MAPKAP kinase-2 and c-Raf in 293 cells. Cells were incubated for 18 h in DMEM containing 0.5% FCS prior to treatment for 60 min with the indicated concentrations of SB 203580. (a) eect of SB 203580 on MAPKAP kinase-2 measured as a percentage of the activity observed in the absence of the drug; (b) eect of SB 203580 on cRaf activity measured as fold activation relative to that obtained in the absence of the drug. MAPKAP kinase-2 and c-Raf were assayed after immunoprecipitation from cell lysates and the results are presented as mean+s.e.m for three separate experiments The activation of c-Raf by SB 203580 is not caused by interaction of the drug with SAPK2/p38a Since SB 203580 inhibits SAPK2a/p38a and SAPK2b/ p38b (collectively termed SAPK2/p38), as well as cRaf, the activation of c-Raf by SB 203580 could have resulted from the binding of this drug to SAPK2/p38, rather than c-Raf. If SB 203580 exerted its eects via interaction with SAPK2/p38, then signals that activate SAPK2/p38 might have been expected to suppress the activation of c-Raf by growth factors. However, neither osmotic shock, UV-C irradiation or the protein synthesis inhibitor anisomycin, which are all potent activators of SAPK2/p38 in Swiss 3T3 and other cells, have any eect on the EGF-induced activation of MAPK2 (Doza et al., 1998). Figure 7 Eect of transfection with an SB 203580-insensitive mutant of SAPK2a/p38 mutant on the activation of MAPKAP kinase-2 and c-Raf by SB 203580. 293 cells were transfected with Thr 106Met SAPK2a/p38a (hatched bars) or mock-transfected (®lled bars). The cells were then incubated for 18 h in DMEM containing 0.5% FCS prior to treatment for 60 min with (+) or without (7) 1 mM SB 203580. (a) Eect of SB 203580 on MAPKAP kinase-2 activity; (b) eect of SB 203580 on c-Raf activity. The protein kinases were assayed after their immunoprecipitation from the cell lysates and the results are presented as mean+s.d. for a representative experiment. Similar results were obtained in one other experiment MAPKAP kinase-2, an immediate downstream target of SAPK2/p38, is partially active in unstimulated 293 cells, and maximal suppression of this basal level of activation is observed when the cells are ®rst preincubated with 1 mM SB 203580 (Figure 6a). In contrast, 1 mM SB 203580 usually induces about a threefold activation of c-Raf and activation rises to 16-fold as the SB 203580 concentration is increased to 10 mM (Figure 6b). These results are consistent with the activation of MAPKAP kinase-2 being blocked by the interaction of SB 203580 with SAPK2/p38 and with c-Raf activation being triggered by the interaction of SB 203580 with another Effect of SB203580 on Raf CA Hall-Jackson et al 2052 protein that binds to the drug with lower anity (presumably c-Raf itself). In order to obtain further evidence that the activation of c-Raf by SB 203580 results from its interaction with c-Raf, and not with SAPK2/p38, we transfected 293 cells with a SAPK2a/p38 mutant in which Thr 106 had been changed to Met to make it insensitive to low concentrations of SB 203580 (Wilson et al., 1997; Eyers et al., 1998). In cells transfected with this mutant, MAPKAP kinase-2 activity is elevated, but can no longer be suppressed by 1 mM SB 203580 in contrast to untransfected cells (Figure 7a). In contrast, basal c-Raf activity increased several-fold after transfection with the Thr 106Met mutant, but c-Raf could still be activated further by incubating the cells with SB 203580 (Figure 7b). Discussion In this paper we have shown that SB 203580 inhibits cRaf (Figure 1) and B-Raf (results not shown) at micromolar concentrations, consistent with the sensitivity to this drug being determined by the presence of threonine (or a smaller residue) at the position equivalent to Thr 106 of SAPK2a/p38. The sensitivity of c-Raf to SB 203580 was 10-fold greater if the enzyme was assayed directly in Sf9 cell extracts than when assayed after immunoprecipitation (Figure 1). This suggests that the interaction of c-Raf with the anti-c-Raf antibody decreases the anity of c-Raf for the drug. An alternative explanation is that the sensitivity of c-Raf to the drug is enhanced by its interaction with a protein(s) that is present in the extracts but removed by immunoprecipitation. The latter explanation seems less likely because the c-Raf assay employed is extremely sensitive and the Sf9 cell extracts have to be diluted 30 000-fold to ensure that initial rate conditions are met. However, whichever of the two explanations is correct, the IC50 value measured without immunoprecipitation is more likely to re¯ect the situation in intact cells. SB 203580 does not prevent the activation of MKK1 or MAPK2 by a variety of growth factors or phorbol esters in many cells (e.g. Figure 2). One possible explanation for these ®ndings is that the activity of Raf isoforms are not rate-limiting for agonist-induced activation of the MAP kinase cascade in the cells that we have studied, and that, in the presence of SB 203580, another growth factor/phorbol ester-stimulated protein kinase(s) mediates the activation of MKK1. However, another explanation is suggested by the observation that incubation of mammalian cells with SB 203580 induces a remarkable activation of c-Raf (when measured in the absence of the drug). Thus Raf inhibition may be counterbalanced by its reactivation. This eect is not mediated by the interaction of SB 203580 with SAPK2a/p38a (Figure 7b), but with another protein that binds to SB 203580 with lower anity (Figure 6b). This protein is likely to be Raf itself, because exactly the same phenomenon is observed with another inhibitor of c-Raf that is structurally unrelated to SB203580 (Clare Hall-Jackson, Philip Cohen and Philip Hedge, unpublished work). The activation of c-Raf by two structurally unrelated c-Raf inhibitors suggests that, in the absence of growth factors/phorbol esters, a mechanism may exist by which c-Raf suppresses its own activation. This putative feedback loop would have to involve a novel pathway because it is unaected by inhibitors of protein kinase C, PtdIns 3-kinase or the MAP kinase cascade (Figure 4), and does not involve changes in the GTP loading of Ras (Figure 5). However, other interpretations are possible. For example, the interaction of these drugs with c-Raf may cause c-Raf to oligomerise (Farrar et al., 1996; Luo et al., 1996) or to be targetted to the plasma membrane (Leevers et al., 1994; Stokoe et al., 1994), which are well-established mechanisms for inducing the activation of c-Raf. It is also curious that incubation of Swiss 3T3 cells for 60 min with SB 203580 does not trigger any activation of MAPK2 and yet subsequent exposure to EGF (in the continued presence of SB 203580), which only increases c-Raf activity a further 3 ± 4-fold (Figure 3a), triggers essentially complete activation of MAPK2 (Figure 3b, Alessi et al., 1995a). This observation could be explained if Raf activity was not rate-limiting for the activation of MKK1 in these cells or if, in addition to activating c-Raf per se EGF makes c-Raf competent to activate MKK1 in vivo by facilitating the interaction of these protein kinases in an unknown way. Many oncogenes and tumour promoters are thought to induce cell transformation, at least in part, by causing inappropriate activation of the MAP kinase cascade. Consistent with this view, the drug PD 98059 which interacts with MKK1 and prevents its activation in vivo, reverses the transformed phenotype of several Ras-transformed cell lines. However, SB 203580, as well as another c-Raf inhibitor that bears no structural relationship to SB 203580, fails to prevent growth factor or phorbol ester-induced activation of MAP kinase. The latter inhibitor also fails to reverse the phenotype of Ras-transformed cell lines (P Hedge, unpublished work). These observations suggest that inhibitors of the kinase activity of Raf may not be eective as anti-cancer drugs. Materials and methods Materials Ro 318220 was a kind gift from Dr D Bradshaw (Roche Pharmaceutical Company, Welwyn Garden City, UK), PD 98059 and SB 203580 were purchased from Calbiochem and wortmannin and phorbol 12, 13 myristate acetate (PMA) from Sigma. These drugs were dissolved in dimethyl sulphoxide (DMSO) at concentrations of 10 ± 50 mM. They were either diluted appropriately in aqueous buers just prior to use, or added directly to the cell culture media to achieve ®nal concentrations of 100 nM ± 50 mM. Control experiments contained the equivalent amounts of DMSO which did not exceed 0.2% (v/v) in any experiment. This concentration did not aect the activity or activation of any protein kinase examined. Epidermal growth factor (EGF) was purchased from Boehringer Mannheim and the monoclonal anti-H-Ras antibody (Y13 259) from Oncogene Science Products. The antibodies against c-Raf (Alessi et al., 1995b), MAPKAP kinase-2 (Clifton et al., 1996), MAP kinase kinase-1 (MKK1) and MAPK2 (also called ERK2) were raised in sheep at the Scottish Antibody Production Unit (Carluke, Lanarkshire, UK). The anti-MKK1 antibodies were raised against the peptide PKKKPTPIQLNPAPDG correspond- Effect of SB203580 on Raf CA Hall-Jackson et al ing to residues 2 ± 17 and the anti-MAPK2 antibodies against the C-terminal peptide ± EETARFQPGYRS. All peptides were conjugated to both bovine serum albumin and keyhole limpet haemocyanin before injection into the sheep. Cell culture and stimulation Con¯uent mouse Swiss 3T3 ®broblasts or human 293 cells were incubated for 18 h in Dulbecco's modi®ed Eagles medium (DMEM) containing 0.5% FCS. After treatment with growth factors or inhibitors as indicated, each 10-cm dish of cells was lysed in 0.4 ml of ice-cold Buer A (20 mM Tris acetate, pH 7.5, 0.27 M sucrose, 1% (by mass) Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium ¯uoride, 10 mM sodium b-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 0.1% (by vol) 2-mercaptoethanol, 0.1 mM phenylmethylsulphonyl ¯uoride, 1 mM benzamidine, 5 mg/ml leupeptin), and the lysates frozen immediately in liquid nitrogen and stored at 7808C until use. Protein concentrations were determined by the method of Bradford (1976). Immunoprecipitation of protein kinases c-Raf was immunoprecipitated from 400 mg cell lysate protein, MKK1 from 100 mg of cell lysate and MAPK2 and MAPKAP kinase-2 from 50 mg cell lysate protein using 5 ml of protein GSepharose conjugated to 4 mg of the appropriate antibody (Alessi et al., 1995b; Clifton et al., 1996). Assay of protein kinases c-Raf, MKK1 and MAPK2 (Alessi et al., 1995b) and MAPKAP kinase-2 (Clifton et al., 1996) were assayed as described previously. c-Raf activity was measured in a coupled assay containing MKK1, MAPK2 and its substrate myelin basic protein (MBP). One unit of c-Raf activity was that amount which increased the activity of MAPK2 by 1 unit/min. MKK1 was assayed by the activation of bacterially expressed MAPK2 (Alessi et al., 1995b). One unit of MKK1 activity was that amount which increased the activity of MAPK2 by 1 unit/min. One unit of MAPK activity was that amount which catalysed the phosphorylation of 1 nmol of MBP in one min. Measurement of guanine nucleotides bound to Ras Confluent Swiss 3T3 cells were incubated for 18 h in DMEM containing 0.5% FCS, then for 1 h in phosphate-free DMEM before labelling for 4 h with 32P-orthophosphate (5 mCi per 10-cm dish). After stimulation, cells were washed with ice-cold 20 mM Tris/HCl pH 7.5, 150 mM NaCl and lysed in Buer B (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mg/ml Bovine Serum Albumin (BSA), 1 mM sodium phosphate, complete proteinase inhibitor cocktail (Boehringer Mannheim), 100 mM GTP, 100 mM GDP, 1 mM ATP) containing 1% (by mass) Triton X-114 (Boehringer Mannheim). Nuclei were removed by centrifugation for 5 min at 13 000 g, and the supernatant incubated for 3 min at 378C and centrifuged for 2 min at 13 000 g to separate the Triton X-114 and aqueous phases. The detergent phase was diluted 10-fold with Buer B plus 0.5 M NaCl. The lysate was precleared with protein G-Sepharose coupled to rabbit pre-immune serum (15 min) and Ras immunoprecipitated using 25 ml protein G-Sepharose beads coupled to 10 mg anti-H-Ras antibody (60 min). The immunoprecipitates were washed eight times with Buer C (50 mM HEPES, pH 7.4, 500 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, 0.005% SDS). GTP and GDP were eluted by incubation for 2 min at 708C in 2 mM EDTA, 2 mM DTT, 0.2% (by mass) SDS, 0.5 mM GDP, 0.5 mM GTP and separated on polyethyleneimine (PEI) cellulose plates (Merck) developed in 1.2 M ammonium formate, 0.8 M HCl. Plates were autoradiographed and the radioactive spots cut out and analysed by Cerenkov counting (Burgering et al., 1991). Immunoblot analysis Cell lysates (7.5 mg protein) were separated on 10% polyacrylamide gels and immunoblotted using 2 mg/ml anti-c-Raf antibody. SAPK2a/p38a expression vectors Human SAPK2a/p38a (CSBP2 isoform) and Thr106Met SAPK2a/p38a with a haemaglutinnin (HA)-tag (YPYDVPDYA) at the amino terminus were produced by PCR ampli®cation, using the previously described constructs (Eyers et al., 1998) as templates. Following DNA sequencing, the ampli®ed DNAs were subcloned into the eukaryotic vector pCMV5 (Invitrogen). 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