Oncogene (1999) 18, 1391 ± 1400 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc Cot protooncoprotein activates the dual speci®city kinases MEK-1 and SEK-1 and induces dierentiation of PC12 cells
Dorthe Hagemann, Jakob Troppmair and Ulf R Rapp
Institut fuÈr la Medizinische Strahlenkunde und Zellforschung (MSZ), University of WuÈrzburg, Versbacher Str. 5, 97078 WuÈrzburg, Germany
Mitogenic signals initiated at the plasma membrane are phosphorylation by the serine/threonine kinase c-Raf- transmitted to the nucleus through an intricate signalling 1 (Daum et al., 1994). The activation of c-Raf-1 network. We identi®ed the protooncoprotein Cot as a following growth factor receptor stimulation typically new component of mitogenic signalling cascades, which involves translocation to the plasma membrane, which activates both the classic cytoplasmic cascade and the is mediated by association of c-Raf-1 with GTP bound SAPK stress pathway. Wildtype and activated Cot Ras (Avruch et al., 1994; Daum et al., 1994). phosphorylate and activate MEK-1 and SEK-1 in vitro. A second well characterized mammalian protein These ®ndings are consistent with the sequence homology kinase cascade which is activated after stress such as between Cot and the rat gene Tpl-2. Expression of heat shock, UV-irradiation, osmotic stress or certain oncogenic Cot in 293, NIH3T3 and PC12 cells leads to cytokines and activation of G protein coupled in vivo phosphorylation of endogenous c-Jun and Erk-1/2 receptors leads to the activation of JNK/SAPK (Hibi suggesting that the serine/threonine kinase Cot functions et al., 1993; Kyriakis et al., 1994; Woodgett et al., beside c-Raf-1 and Mos as a direct activator of MEK-1. 1996). JNK/SAPK is phosphorylated and activated by Furthermore, we have examined the biological eects of the dual speci®city kinase SEK/MKK4 (Davis, 1994; Cot on the phenotype of ®broblastic and neuronal cells. Robinson and Cobb, 1997). Recently, a third In order to test a potential c-Raf-1 dependency of Cot mammalian MAPK has been cloned: p38 or RK. p38 transformation, the eect of oncogenic Cot on Raf is phosphorylated and activated by MKK3, MKK6 revertant CHP25 cells was determined. Cot could restore and in overexpression experiments also by MKK4 the transformed phenotype indicating that Cot transfor- (Davis, 1994; Han et al., 1994; Robinson and Cobb, mation is not dependent on active c-Raf-1 and that Cot is 1997). not a target for the putative Raf inhibitor, which is The serine/threonine kinase Cot was originally presumably active in the revertant cell line. Expression of cloned from embryonic SHOK cells transformed with oncogenic versions of Raf as well as v-Mos leads to DNA extracted from a human thyroid carcinoma cell dierentiation of PC12 cells. Cot also induces neurite line (Miyoshi et al., 1991). The cot gene has suered a outgrowth of PC12 cells. These data are consistent with gene rearrangement within the last coding exon of the the role of Cot in the classic mitogenic cascade and cot proto-oncogene during this procedure (Miyoshi et suggest that the simultaneously activated JNK/SAPK al., 1991). The ®rst 397 amino acids of the Cot protein stress pathway has no antagonistic eects in this context. are identical to the oncogenic Cot (CotDC), whereas the 70 carboxyterminal amino acids are replaced by 17 Keywords: cot; classic cytoplasmic cascade; stress completely dierent ones (Aoki et al., 1993). Cotrans- kinase pathway; transformation; dierentiation fection experiments with the oncogenic cot and HA- ERK-1 in 293 cells showed activation of ERK-1, which was sensitive to inhibition by the dominant negative c- Raf-1 mutant C4 (Troppmair et al., 1994). Further Introduction experiments revealed that transformation of NIH3T3 cells by v-cot resulted in a hyperphosphorylated, Activation of growth factor receptors stimulates a shifted form of c-Raf-1, which is typical for the active variety of parallel signal transduction cascades kinase. These results suggested functional interaction (reviewed in Daum et al., 1994; Slupsky et al., 1998). between Cot and c-Raf-1 (Troppmair et al., 1994). The best understood signalling pathway is the classic Cot and Tpl-2 are highly related at the amino acid cytoplasmic cascade, which regulates cell proliferation level (Miyoshi et al., 1991). Tpl-2 was originally and dierentiation and leads to activation of ERK-1 identi®ed as the product of an oncogene involved in and ERK-2 (Rapp, 1991; Blenis, 1993; Daum et al., the progression of Moloney murine leukemia virus- 1994; Slupsky et al., 1998). Mammalian ERKs are induced T-cell lymphomas in rats (Patriotis et al., activated by phosphorylation on a threonine and a 1993). Tpl-2 is an intergration locus of the provirus, tyrosine residue in a conserved TEY motif by the dual which leads to expression of a truncated mRNA and speci®city protein kinases MEK-1 and MEK-2 (Crews an activated kinase, that is altered at the C-terminus and Erikson, 1992; Cano and Mahadevan, 1995). (Makris et al., 1993). Overexpression of Tpl-2 induces These kinases are activated in turn through ERK activation in COS-1 and NIH3T3 cells and this activation may depend on the cooperative action of Tpl-2, Ras and c-Raf-1 in a multimolecular complex that leads to the phosphorylation of MEK-1 (Patriotis Correspondence: UR Rapp Received 5 June 1998; revised 16 September 1998; accepted 16 et al., 1994). Recently it has been reported that Tpl-2 September 1998 can phosphorylate and activate MEK-1 and SEK-1 in Activation of MEK-1 and SEK-1 by Cot D Hagemann et al 1392 COS-1 and Jurkat cells independent of Ras and c-Raf- tated from Sf9 or 293 cells and tested for their ability 1 (Salmero n et al,. 1996). to phosphorylate kinase inactive GST ± MEK as a In this study we have examined the biochemical and substrate in vitro. As shown in Figure 1c Cot biological function of Cot in terms of eects on protein phosphorylates MEK. The level of MEK phosphoryla- kinase cascades that regulate growth as well as the tion obtained with CotDC was considerably greater phenotype of ®broblastic and neuronal cells. Specifi- than that for Cot wildtype even though the amount of cally, we show that Cot is involved in the classic immunoprecipitated CotDC as determined by Western cytoplasmic cascade and the SAPK stress pathway. It blotting was lower than the amount of wildtype Cot. phosphorylates and activates both MEK-1 and SEK-1, To test directly whether the in vitro phosphorylation and induces by this ERK and SAPK activation, in vitro of MEK-1 by Cot resulted in its activation a coupled and in vivo. These properties are consistent with the assay was used. Cot immunoprecipitates (Figure 1d) sequence homology between cot and the rat gene tpl-2, were assayed for their ability to phosphorylate and suggesting that these kinases are encoded by cognate activate wildtype GST ± MEK-1 by including ERK-1 as genes. Overexpression of oncogenic Raf or Mos, which substrate in the kinase reaction. As shown in Figure are also known to activate MEK, leads to differentia- 1d, MEK-1 was activated after incubation with tion of PC12 pheochromocytoma cells dependent on immunoprecipitated wildtype or oncogenic Cot as ERK activation (Xia et al., 1995; Wixler et al., 1996). well as with immunoprecipitated active c-Raf-1 which We examined whether the oncogenic version of cot was was used as a positive control. The oncogenic form of able to induce neuronal dierentiation of PC12 cells. Cot had a 4.5-fold higher kinase activity compared to Indeed, this constitutively active MEK activator Cot the wildtype form. leads to neurite outgrowth. Furthermore we showed Both ERK and SAPK can phosphorylate and thus that Cot could restore the transformed phenotype of modulate the activity of transcription factors. Raf revertant cells (Kolch et al., 1993) indicating Reporter gene assays were thus used to further independence from the active c-Raf-1 kinase. analyse Cot mediated signalling pathways. The reporter construct pB4x (Bruder et al., 1992) contains four copies of the oncogene responsive element from the polyomavirus enhancer inserted upstream of the Results minimal b-globin promoter fused to the luciferase (luc) gene. It is known that expression of activated Cot stimulates ERK via phosphorylation and activation Raf stimulates this promoter, which contains Ap-1- of MEK and Ets-binding sites in NIH3T3 cells (Bruder et al., To analyse the eect of Cot on the activation of ERK, 1992). Transfection experiments in 293 cells revealed 293 cells were transfected with wildtype, oncogenic, or that oncogenic as well as wildtype Cot were very a kinase dead form of cot alone or in combination with strong activators of this promoter (Figure 4a). The HA-tagged ERK-1-DNA (Figure 1a). Forty-eight measured luciferase activity was twofold higher than hours after transfection the cells were lysed and the activity induced by an equal amount of c-Raf-1- ERK-1 was immunoprecipitated and assayed for its BXB DNA. To analyse whether this Cot mediated ability to phosphorylate MBP. Coexpression of ERK-1 promoter activation is dependent on the ERK with c-Raf-BXB, an activated form of c-Raf-1 (Bruder pathway, 293 cells were transfected with a dominant et al., 1992), was used as a positive control. In negative mutant of ERK (ERKB3 K52/R52) (Robbins agreement with our previously published data we et al., 1993) in combination with Cot and the showed that oncogenic Cot activates ERK-1. Addi- reporter-construct. The mutant was able to inhibit tionally we demonstrated that overexpression of the Cot mediated promoter activation by 80% wildtype Cot also activated ERK-1, whereas a kinase suggesting that the ERK pathway was required for inactive mutant of Cot failed to do so. the Cot mediated activation of the Ap-1- and Ets- To test at which level of the classic cytoplasmic driven promoter (Figure 4a). cascade Cot functions, Sf9 cells were infected with the baculovirus expression constructs for wildtype or Cot stimulates SAPK via SEK phosphorylation but has oncogenic forms of Cot alone or in combination with no eect on p38 recombinant MEK-1 viruses (Figure 1b). Forty-eight hours after infection, MEK-1 was immunoprecipitated To examine the involvement of Cot in other signal and its kinase activity was assayed using GST ± ERK-1 transduction pathways, 293 cells were co-transfected as a substrate. Expression of either the wildtype or the with plasmids encoding Cot and HA-tagged SAPKb oncogenic form of Cot resulted in strong activation of (Figure 2a, left panel). Forty-eight hours after MEK-1. No dierence between both forms of Cot was transfection, SAPKb was immunoprecipitated and seen when the protein concentrations were taken into tested for its ability to phosphorylate GST-c-Jun consideration. This could be due to the rate limiting fusion protein. Expression of either the wildtype or amount of immunoprecipitated MEK-1. This suspicion the oncogenic form of Cot resulted in a strong was supported by ®ndings in Sf9 cells where a twofold activation of SAPKb. increase of MEK-1 expression revealed dierences In order to test at which level of the cascade Cot between oncogenic and wildtype Cot in their ability triggers the activation of SAPK, 293 cells were to activate MEK. These results demonstrate that the transfected with the wildtype or the oncogenic form Cot induced ERK activation involves MEK and that of Cot together with SAPKb and a kinase-inactive wildtype Cot can be activated by overexpression. mutant of SEK (Sanchez et al., 1994; Ludwig et al., To directly investigate whether MEK is a substrate 1996). As demonstrated in Figure 2a (right panel) an for Cot, wildtype Cot or CotDC was immunoprecipi- excess of dominant negative SEK was able to block the Activation of MEK-1 and SEK-1 by Cot D Hagemann et al 1393 Cot-induced SAPK activation suggesting a function of a direct substrate for Cot whereas no SEK phosphory- Cot upstream or at the level of SEK. lation was seen with immunoprecipitates of activated To test whether Cot can directly phosphorylate Raf-1 (data not shown). SEK, immunoprecipitated oncogenic Cot was tested To analyse whether the SAPK pathway is involved for its ability to phosphorylate kinase inactive GST- in the Cot mediated Ap-1 and Ets driven promoter SEK K167R in vitro. As shown in Figure 2b, SEK was activation, 293 cells were transfected with a dominant
a b
c d
Figure 1 Cot stimulates ERK via activation of MEK-1 by direct phosphorylation. Lysates were prepared from 293 cells 48 h after transfection with dierent cot constructs alone or in combination with ERK-1 (a). ERK-1 was immunoprecipitated from cell lysates and assayed for its ability to phosphorylate MBP in vitro. Overexpression of CotDC and Cotwt but not of the kinase dead mutant of CotDC led to ERK-1 activation (upper panel). Coexpression of c-Raf-BxB was used as a positive control. Western blot analysis con®rmed the expression of the transfected genes. The expression of Cot in the cotransfection experiment together with ERK was low and only seen in a longer exposure resulting in the appearance of an unspeci®c 60 kDa which runs slightly below wildtype Cot. To observe the MEK-1 dependency of Cot induced ERK-1 activation Sf9 cells were infected with recombinant baculoviruses containing wildtype or oncogenic cot alone or in combination with recombinant MEK-1-viruses (b). MEK-1 was immunoprecipitated from cell lysates and assayed for its ability to phosphorylate kinase dead GST-ERK-1 in vitro. Overexpression of CotDC and Cotwt led to MEK-1 activation. Western blot analysis con®rmed the expression of the transfected genes. Results shown have been normalized to the activity or immunoprecipitated MEK-1 from lysates or uninfected Sf9 cells, which was arbitrarily chosen as one. Data represent the mean (+ standard error) of six assays. Direct GST-MEK-1 phosphorylation by oncogenic and wildtype Cot was observed by immunoprecipitating the overexpressed kinases from Sf9- or 293 cell lysates (c). The expression of Cot was con®rmed by Western blot analysis (lower panel). Using GST-MEK-1 and a kinase inactive GST-ERK-1 in combination as substrates (coupled assay) for overexpressed and immunoprecipitated Cotwt or CotDC, revealed Cot induced MEK- 1 activation in Sf9 and 293 cells (d). Active c-Raf-1 was used as a positive control. The amount of Cot in immunoprecipitates was determined by Western blotting Activation of MEK-1 and SEK-1 by Cot D Hagemann et al 1394 negative mutant of SEK (K167R) (Sanchez et al., 1994; member of the MAPK family (Davis, 1994; Robinson Ludwig et al., 1996) together with Cot and the and Cobb, 1997). In order to test whether p38 is reporter-construct. The mutant was able to inhibit activated by Cot, 293 cells were cotransfected with Cot the Cot mediated promoter activation by 80% and p38 (Figure 2c). Forty-eight hours after transfec- suggesting that the SAPK pathway was required as tion p38 was immunoprecipitated and assayed for its well for the Cot mediated activation of the Ap-1- and ability to phosphorylate 3pK, a known substrate of Ets-driven promoter (Figure 4a). p38 in vitro (Ludwig et al., 1996). p38 transfected cells A second protein kinase cascade activated by stress stimulated with anisomycin were used as a positive stimuli leads to activation of p38 kinase, another control. As shown in Figure 2c Cot did not activate
a b
c
Figure 2 Cot stimulates SAPK activation by direct SEK phosphorylation but has no eect on p38 activation. (a) Lysates were prepared from 293 cells 48 h after transfection with dierent cot constructs alone or in combination with SAPKb (left panel). SAPKb was immunoprecipitated from cell lysates and assayed for its ability to phosphorylate c-Jun in vitro. Overexpression of CotDC and Cotwt led to SAPKb activation (upper panel). Cotransfected kinase dead SEK blocked the Cot induced SAPK activation (right panel). Western blot analysis con®rmed the expression of the transfected genes. (b) Direct GST-SEK phosphorylation by oncogenic and wildtype Cot was observed by immunoprecipitating the overexpressed kinases from 293 cell lysates. The expression of Cot was con®rmed by Western blot analysis (lower panel). (c) 293 cells were transfected with the vectors indicated, p38 was puri®ed from cell lysates by immunoprecipitation and tested for its ability to phosphorylate GST-3pK in vitro. The expression of Cot and p38 was con®rmed by Western blot analysis (lower panels). Anisomycin stimulated cells were used as a positive control. No Cot induced p38 activation was detectable. The Cot induced downregulation of p38 kinase activity is not signi®cant taken together the results of three independent kinase assays Activation of MEK-1 and SEK-1 by Cot D Hagemann et al 1395 terminally truncated form of oncogenic cot (CotDCD) without the oncogene speci®c sequence was used to exclude an in¯uence of this part of the gene. This deletion mutant showed a 30% higher luciferase activity in comparison to CotDC, indicating that the C-terminus may be involved in negative regulation of the kinase. As shown in Figure 4b a dominant negative mutant of Cot could substantially block the c-Raf-1-BXB mediated promoter activation. The dominant negative mutant c- Raf-C4B (Bruder et al., 1992), which interferes with Ras-dependent activation of c-Raf-1, reduced the CotDCD induced promoter activation about 90%. These ®ndings con®rmed the model of a cooperative action of the two MEK activators but stood in contrast Figure 3 Phosphorylation of ERK1/2 and c-Jun by Cot in vivo. to the transformation assay in CHP25 cells. Further- 293 cells were transfected with the oncogenic Cot or stimulated more this data raised the possibility that regulators of with TPA or anisomycin as indicated. Cell lysates were analysed Raf might aect Cot activity. by phosphospeci®c ERK or c-Jun antibodies (upper panel) and the expression of Cot and endogenous ERK and c-Jun was con®rmed by Western blot analysis (lower panels). Overexpressed Cot induces dierentiation of PC12 cells Cot induces an ERK1/2 as well as a c-Jun phosphorylation in vivo PC12 pheochromocytoma cells have been used as a model system for dierentiation of neuronal cells. Observations in PC12 cells gave evidence that the p38 nor did transfection of wildtype, activated or dynamic balance between ERK- and SAPK/p38 oncogenic Cot interfere with anisomycin stimulation of pathways is important in determining whether a cell p38 activity (data not shown). survives or undergoes apoptosis (Xia et al., 1995). After stimulation with NGF, which activates the classic cytoplasmic cascade, the cells stop growing and start to Cot induces ERK1/2 and c-Jun phosphorylation in 293 dierentiate. NGF withdrawal led to sustained cells activation of the SAPK and p38 kinases and The ability of cot to stimulate MAPK in vivo was inhibition of ERKs (Xia et al., 1995). Our data tested by transfection of 293 cells with oncogenic Cot showed that Cot activates both the classic cascade as or stimulation with either TPA or anisomycin (Figure well as the SAPK pathway. To analyse whether Cot 3). Forty-eight hours after transfection phosphorylated induces apoptosis or dierentiation, PC12 cells were ERK and c-Jun were detected in the lysates using a infected with retrovirus expressing the oncogenic form phospho-ERK1/2 or phospho-c-Jun speci®c antibody. of Cot. After 5 days the cells showed a dierentiated Overexpression of oncogenic Cot induces in 293 cells morphology like the NGF stimulated cells, which were phosphorylation of endogenous ERK1/2 as well as of used as a positive control (Figure 6). Thus, like the c-Jun. MEK activators c-Raf-1 and Mos, Cot also induced neurite outgrowth in PC12 cells. Cot reverts CHP25 cells Cot stimulates ERK and JNK activation in PC12 and The truncated Cot has been shown to have transform- NIH3T3 cells ing activity in hamster SHOK cells as well as in NIH3T3 ®broblasts (Miyoshi et al., 1991). Our data Expression of oncogenic Cot results in the transforma- indicated that Cot is a MEK activator and functions in tion of NIH3T3 cells and induces neurite outgrowth in the classic cytoplasmic cascade on the same level as c- PC12 cells. In order to analyse which MAPK pathways Raf-1. We have previously reported that Cot induced are triggered in these cells the activation status of ERK ERK-1 activation was sensitive to inhibition by a and JNK was determined using phospho-ERK and dominant negative c-Raf-1 mutant (Troppmair et al., phospho-Jun speci®c antibodies (Figure 7). ERK1/2 1994). To test the c-Raf-1 dependency of the was strongly phosphorylated in PC12 cells as a result transforming potential of oncogenic Cot we examined of Cot expression. In the case of c-Jun both the the sensitivity of CHP25 cells to transformation by Cot. expression level and the phosphorylation of endogen- CHP25 is a revertant cell line derived from v-raf ous c-Jun are increased. In stably transfected NIH3T3 transformed NIH3T3 ®broblasts, which express a cells oncogenic Cot mediated phosphorylation of functional v-raf gene, but fail to form colonies in soft ERK1/2 and c-Jun with no changes in c-Jun agar and are non tumorigenic (Kolch et al., 1993). As expression levels. Thus in agreement with the 293 cell shown in Figure 5 infection of CHP25 cells with the data (Figure 3) Cot expression results in the in vivo constitutively active form of Cot enabled them to grow activation of JNK and ERK in a variety of cells of in soft agar. The block in CHP25 cells was not extended dierent origin. to Cot. This data demonstrate that Cot worked independently of an active c-Raf-1 kinase or was not eected by an Raf-speci®c inhibitor which may be Discussion active in the revertant cell line. Reporter-gene assays were used to further explore the relationship between c- A rapidly increasing number of kinases were found to Raf-1 and Cot (Figure 4b). For these assays a C- be involved in activation of MAPK cascades, as Activation of MEK-1 and SEK-1 by Cot D Hagemann et al 1396 reported recently (Robinson and Cobb, 1997). The ®rst in vitro, but most potently activates the JNK pathway known MEK activator was c-Raf-1, which is not via SEK phosphorylation. MEKK 2 and 3 are also known to activate MEKs other than MEK1/2 (Rapp et able to signal to both JNK and ERK (Robinson and al., 1983a,b, 1988a,b; Slupsky et al., 1998). MEKK 1 Cobb, 1997). Several other PSKs have been shown to was identi®ed as a kinase which phosphorylates MEK activate the JNK pathway in various cell types (Fanger
Figure 4 AP-1 and Ets transactivation activities of Cot. 293 cells were cotransfected with 0.5 mg of pB4X, 0.5 mg of cot and 5 mg each of the indicated expression vectors. Dominant negative mutants of ERK and SEK partially block the Cot induced transactivation (a). A dominant negative mutant of Cot inhibits the c-Raf-1-BxB mediated promoter activation and a dominant negative c-Raf-1 C4B mutant blocks the Cot induced transactivation (b). The level of luciferase expression was determined from cell extracts harvested 48 h post-transfection. Results shown have been normalized to the activity of transfected empty expression vector (vector control), which was arbitrarily chosen as 1. The standard deviation of the mean values is indicated by error-bars Activation of MEK-1 and SEK-1 by Cot D Hagemann et al 1397
Figure 5 Transformation of Raf revertant CHP 25 cells by Cot. Figure 7 ERK1/2 and JNK activation in Cot expressing PC12 CHP 25 cells were infected with retroviruses expressing the and NIH3T3 cells. PC12 cells were infected with retroviruses oncogenic form of Cot (CotDC). Cot-virus infected and expressing either constitutively active Cot (pJJ26) or c-Raf-1 uninfected NIH3T3 cells were used as controls. Uninfected (EHneo). In the case of Cot two dierent virus dilutions (100, NIH3T3 and CHP25 cells show an untransformed morphology. 1071) were used. Forty-eight hours after infection the cells were The soft agar colony induction by Cot was indistinguishable lysed and analysed for the presence of phosphorylated ERK1/2 or between CHP25 and the parental NIH3T3 cells. Pictures were c-Jun. The expression of endogenous ERK and c-Jun was taken at day 14 after infection con®rmed by Western blot analysis. In the case of NIH3T3 cells stable transfectants expressing the same retroviral Cot construct were used. For PC12 cells the eciency of virus transfer was determined 120 h after infection by monitoring neurite outgrowth. 90% of the Cot and 60% of the EHneo infected PC12 cells were dierentiated. In the case of NIH3T3 cells complete morpholo- gical transformation was used as an indicator for the presence of activated Cot
Most onco-protein kinases are activators of the Ras- Raf-MEK-ERK cascade (Heidecker et al., 1989; Rapp Figure 6 Expression of oncogenic Cot induces neurite outgrowth et al., 1988a, 1994). The unrelated kinases c-Raf-1 and in PC12 cells. PC12 cells were infected with 26106 c.f.u./ml of Mos for example phosphorylate and activate MEK and retrovirus containing a cDNA encoding the constitutively active this MEK activation is required for cell transformation form of Cot (CotDC). Uninfected cells were used as negative and and dierentiation (Troppmair et al., 1994; Rapp et al., NGF stimulated cells were used as positive control. Over- expression of oncogenic cot enhances the dierentiation of 1994; Wixler et al., 1996). Under overexpression PC12 cells. Pictures were taken at day 5 after infection conditions MEKK-1, which functions in the stress kinase pathway, is able to activate ERK-1. However, this kinase cannot transform cells and indeed activa- tion of the SAPK pathway by MEKK-1 inhibits et al., 1997). In this study we present data that growth of NIH3T3 cells (Minden et al., 1994; Yan et demonstrate that the human serine/threonine kinase al., 1994). The ERK and SAPK pathway therefore Cot similarly to the rodent Tpl-2, activates two appear to mediate opposite eects on cell growth when dierent signal transduction cascades namely the activated separately. Furthermore, the stimuli that classic cytoplasmic cascade, leading to the activation trigger ERK and SAPK generally are discrete of ERK, and the stress induced pathway, leading to the suggesting that these pathways are independent (Cano activation of JNK/SAPK. Speci®cally, Cot phosphory- and Mahadevan, 1995). Our results clearly show that lates and activates both MEK-1 and SEK-1 in vitro under overexpression conditions Cot triggers both and induces ERK and c-Jun phosphorylation in vivo. pathways by phosphorylation and activation of MEK However, expression of Cot shows no eect on the and SEK in vitro (Figures 1a and 2c). We extended activation of the second stress induced kinase cascade, these data by the use of phospho-speci®c antibodies in the p38 pathway. Consistent with the eects on ERK vivo (Figures 3 and 7). The Cot induced activation of and SAPK Cot was shown to activate the Ap-1- and immunoprecipitated SAPK could be due to an Ets-driven promoter. Reporter-gene assays revealed an autocrine loop triggered by Cot. Expression of an interdependence between Cot and c-Raf-1. A negative estradiol-dependent form of the c-Raf-1 kinase in regulator of c-Raf-1 that is presumably active in NIH3T3 cells results in constitutive activation of the CHP25 Raf revertant cells has no eect on Cot. In ERK- and the JNK-pathway, but JNK activation is contrast to v-mos, this oncogene transformed CHP25 not observed until 16 ± 24 h after c-Raf-1 activation revertant cells. Furthermore we showed that oncogenic and is independent of de novo protein synthesis Cot had the ability to induce dierentiation of PC12 (McCarthy et al., 1995; Minden et al., 1994). This cells. delayed JNK activation is thought to be mediated by a Activation of MEK-1 and SEK-1 by Cot D Hagemann et al 1398 c-Raf-1 induced autocrine loop involving HB-EGF These observations lead to the model that c-Raf-1 expression, rather than crosstalk between the pathways and Cot may be regulated by the same proteins but in (Kerkho and Rapp, 1997; McCarthy et al., 1995). An a dierent way. Ras is important for the c-Raf-1 overexpression artefact of the Cot induced stimulation activation, but may have suppressor function in Cot of the two kinase cascades cannot be excluded. A signalling. The suppressor activity in CHP25 cells temporal dissociation between Cot induced ERK and seems to be restricted to c-Raf-1 and v-Mos and do JNK activation will be further investigated. It is not aect Cot transforming activity negatively. We will possible that upstream regulators of Cot signalling further analyse whether Cot can compensate Raf in¯uence the phosphorylation of the Cot substrates and function and whether these gene products are thereby determine whether the classic cytoplasmic redundant in the organism. cascade or the JNK pathway is activated. Since Cot is an oncogene and shows transforming Because of the high similarity between the amino activity in NIH3T3 as well as in hamster SHOK cells acid sequence of Cot and Tpl-2 (Miyoshi et al., 1991) (Miyoshi et al., 1991; Aoki et al., 1993) the question and our previous observation of Cot induced ERK-1 arose whether the SAPK/JNK stress cascade, that has activation in 293 cells transiently transfected with Cot been associated with induction of apoptosis, contrib- (Troppmair et al., 1994), it was proposed that Cot may uted to or opposed the oncogenic activity. In stably be the human homologue of the rodent protein transfected NIH3T3-pJJ26 cells both, ERK and c-Jun (Salmero n et al., 1996; Patriotis et al., 1993). Our are phosphorylated whereas the control cells show no results support the notion that Cot and Tpl-2 are phosphorylation (Figure 7). As PC12 cells were cognate genes. Salmero n et al. (1996) recently reported reported to be sensitive to the apoptosis-inducing that Tpl-2 phosphorylates MEK-1 and SEK-1 in vitro. function of JNK/SAPK, we examined the eects of Patriotis et al. (1994) reported that Tpl-2 acts in Cot in this cell system. We observed that oncogenic concert with Ras and c-Raf-1 to activate ERK. Cot also induced neurite outgrowth, indicating the Contrary to this, the data of Salmero n et al. (1996) involvement of Cot in the ERK pathway (Figure 6). indicate that activation of ERK by Tpl-2 was The phosphorylation of endogenous ERK1/2 was independent of both Ras and c-Raf-1. Our data strongly enhanced in Cot infected cells in comparison support the results of Troppmair et al. (1994) and to control cells. In addition both c-Jun phosphoryla- Patriotis et al. (1994). The c-Raf-1 kinase is a key tion and expression were increased (Figure 7). These protein at the entry-point into the classic cascade and results are consistent with recent data reporting that its activation involves multiple interactions of proteins PC12 cell dierentiation requires the induction of c-Jun in a large complex (Daum et al., 1994; Morrison and synthesis as well as c-Jun phosphorylation. Whereas Cutler, 1997; Rapp et al., 1994). Cot and c-Raf-1 are both ERK and JNK can catalyse phosphorylation of interdependent as shown in reporter-gene assays. A the relevant sites in c-Jun, only the former can dominant negative c-Raf-1-mutant (C4B) can partially stimulate c-Jun expression in PC12 cells eciently block the Cot induced promoter activity and a (LeppaÈ et al., 1998). JNK signalling by itself can not dominant negative Cot mutant reduces the c-Raf-1- induce PC12 cell dierentiation (LeppaÈ et al., 1998). BxB induced promoter activity (Figure 4b). These Although not suggested by our experiments it could ®ndings raised the possibility that regulators of c-Raf-1 be that the Cot induced activation of the SAPK/JNK might aect Cot activity. Raf-1-C4B interferes with pathway is cell type dependent. Further experiments Ras-dependent activation of c-Raf-1 suggesting that will be required to understand cooperative versus Ras in¯uences Cot activity as well. Sf9 cells do not antagonistic function of these pathways. express endogenous Ras. Experiments in these cells revealed that Cot is active in the absence of Ras Materials and methods (Figure 1), whereas c-Raf-1 requires coexpression of Ras and a PTK oncogene for full activation (Cai et al., Expression constructs 1997). We have suggestive evidence from experiments that Ras has a negative in¯uence on the Cot kinase Full length Cotwt cDNA was subcloned into pVL1393 for activity (data not shown). Experiments in CHP25 cells baculovirus/Sf9 cell expression experiments and into give further evidence for dierential regulation of Cot pCMV-5 for 293 cell experiments. The oncogenic cotDCc- DNA encodes a c-terminally truncated form of Cot which and Raf as the putative negative regulator of c-Raf-1 terminates at residue 415. An EcoRIfragmentofcotDCc- does not aect Cot. This Raf revertant cell line is DNA was subcloned from pUC13 into the pVL1393 and resistant to transformation by most oncogenes such as pCMV-5. A kinase inactive form of Cot was generated by ras or src which function upstream of c-Raf-1 but were mutating K167 to R in the cotwt-pCMV-5 construct with easily transformed by oncogenic versions of proteins the QuikChangeTM Site-Directed Mutagenesis Kit (Strata- out of the Raf eector pathway such as v-fos (Kolch et gene) using the sense primer 5'-GGCGTGTAGACT- al., 1993). The revertant phenotype of CHP25 cells is GATCCC-3' and the corresponding antisense primer. proposed to result from mutation of a single gene cotDCD, a c-terminal truncated oncogenic form of Cot without oncogene speci®c sequence, was generated with the critical for Raf transformation, for example the loss of TM function of a downstream target of Raf-kinase as QuikChange Site-Directed Mutagenesis Kit (Stratagene) using the sense primer 5'-GATCAGCCACGCTAGGCCC- opposed to the activation of a transdominant CACC-3' and the corresponding antisense primer. The suppressor gene (Kolch et al., 1993). Because the cells mutations were veri®ed by DNA-sequencing. can be retransformed by v-cot (Figure 5) we favour the latter mechanism. As Cot diers from v-Mos, another MEK activator, in its ability to retransform CHP25 Cell lines cells it seems likely that JNK activation by Cot CHP25 cells and NIH3T3 cells were maintained as contributes to transformation of these cells. described by Kolch et al. (1993). Retroviral infections Activation of MEK-1 and SEK-1 by Cot D Hagemann et al 1399 were performed as described previously (Rapp and Todaro, mutant protein (MEK-kinase assay), 4 mg of recombinant 1978). PC12 cells were cultured as described (Wixler et al., K167R kinase dead GST-SEK mutant protein or a kinase- 1996). NGF treatment (Gibco) was performed using a ®nal dead ERK mutant protein and incubated at 308Cfor concentration of 50 ng/ml. No starved cells were used. 15 min. A coupled assay was performed in the presence of Retroviral infections were carried out as described (Rapp 2 mgGST-MEKand2mg of the kinase-dead ERK mutant and Todaro, 1978). (Robbins et al., 1993; Alessi et al., 1994). Samples were Sf9 insect cells were maintained in IPL-41 insect medium resuspended in 30 ml SDS ± PAGE loading buer (60 mM (Gibco) supplemented with 10% heat-inactivated fetal calf Tris/HCl, pH 6.8, 10% (v/v) glycerol, 3% (w/v) SDS, 5% serum (PAN), 1% tryptose, 2 mML-glutamine, 100 U/ml (v/v) b-mercaptoethanol, 0.005% (w/v) bromphenol blue). penicillin/streptomycin. The cells were propagated at 278Cin Following size fractionation in 10% SDS ± PAGE gels, a suspension culture. Infections were performed with 107 Sf9 proteins were transferred to a nitrocellulose membrane cells and 10 MOI of baculoviruses expressing Cotwt, CotDC, using a tank blot procedure (Bio-Rad Mini-PROTEAN II) CotK167R or MEK-1. The cells were cultured at 278C for in a buer containing 25 mM Tris and 190 mM Glycine. 48 h before harvesting. The blot was performed at 400 mA for 1 h. Quanti®cation 293 embryonal kidney cells were grown in Dulbecco's of kinase activity was done using the Bio Imaging Analyser modi®ed Eagle's medium (Gibco) supplemented with 10% BAS 2000 (Fuji). heat-inactivated fetal calf serum (PAA), 2 mML-glutamine, 100 U/ml penicillin/streptomycin. The cells were maintained Immunoblotting at 378C5%CO2 and 90% humidity. Soft agar-cloning was performed as described (Rapp and Todaro, 1978). Transfec- Proteins were separated as described above. The membrane tions were performed following a modi®cation of the calcium was blocked over night at 48C in PBS buer supplemented phosphate coprecipitation method (Chen and Okayama, with 5% (w/v) nonfat dried milk and 0.05% (v/v) Tween 20 1987). Total amount of DNA transfected was kept constant and incubated on a platform shaker at room temperature by ®lling up with vector DNA. Six to twelve hours after for 3 h with appropriate dilutions of the antibodies in transfection precipitates were removed by washing with blocking buer. Afterwards the blot was washed three phosphate buered saline (PBS) and cells were refed with times for ten minutes with PBS buer supplemented with medium containing 0.3% serum. Forty-eight hours after 0.05% Tween 20 and incubated on a platform shaker at transfection cells were washed with PBS and cell pellets were room temperature for 1 h in PBS buer supplemented with either shock frozen in liquid nitrogen and stored at 7808Cor 1.6% (w/v) nonfat dried milk and a 1:3000 dilution of analysed directly. Protein A horseradish peroxidase-coupled secondary anti- body. The blot was washed three times for 10 min with PBS buer supplemented with 0.05% Tween 20 and the Antibodies protein bands were visualized using the ECL detection Immunoprecipitation and Western blotting of Cot and Tpl- system (Amersham). 2 was carried out using the rabbit antiserum Cot-AS 500 raised against a N-terminally peptide corresponding to aminoacids 5 ± 23 and the goat Tpl-2 antiserum N-17 Luciferase assay (Santa Cruz). For Immunoprecipitation and Western Cell pellets were lysed in 400 mlharvestingbuer(50mM blotting the following antibodies were used: c-Raf-1 501, Tris, 50 mM MES, pH 7.8, 1 mM DTT, 0.1% Triton X- rabbit polyclonal (Rapp lab); MEK-1 (C-18), rabbit 100), precleared by centrifugation (15 000 g,10minat polyclonal (Santa Cruz); 12CA5, mouse monoclonal 48C), standardized for protein concentration and 50 mlof (Field et al., 1988); PhosphoPlusTM p44/42 MAPK lysate were analysed together with 50 ml of assay buer (Tyr204), rabbit polyclonal (Biolabs); ERK1 (C16) rabbit (125 mM Tris, 125 mM MES, pH 7.8, 25 mM magnesium polyclonal (Santa Cruz); c-Jun (KM-1) mouse monoclonal acetate, 5 mM ATP) and 50 mlofD-luciferin (1 mM in (Santa Cruz); c-Jun, rabbit polyclonal (Biolabs). 5mM KHPO4, pH 7.8) in a luminometer (Microlumat LB 96P, Berthold AG, Germany). Immunoprecipitation Forty-eight hours after infection (Sf9 cells) or transfection (293 cells) cells were washed twice with ice-cold PBS and lysed in NP-40 buer (0.2% (v/v) NP-40, 25 m Tris/HCl, M Abbreviations pH 7.5, 150 m NaCl, 1 mM DTT, 1 mM EDTA, 1 mM M Cot, cancer osaka thyroid; ERK, extracellular signal EGTA, 20 mM NaF, 1 mM Na-orthovanadate, 10 mM Na- regulated kinase; MEK, MAPK/ERK kinase; MAPK, pyrophosphate, 1 mg/ml aprotinin and 1 mg/ml leupeptin) mitogen activated protein kinase; MKK, MAPK kinase; for 15 min at 48C. The lysates were precleared by SAPK, stress activated protein kinase; JNK, c-Jun N- centrifugation (12 000 g at 48C for 10 min). Nine hundred terminal kinase; PSK, protein serine kinase; PTK, protein mg of total cellular protein were incubated with 20 mlof tyrosine kinase; Tpl-2, tumor progression locus 2; SHOK, Protein G agarose (Boehringer) and the indicated antibody syrian hamster Osaka kanazawa; HA, hemaglutinin; MOI, for Cot or MEK-1 proteins for 2 h at 48C. The multiplicity of infection; NGF, nerve growth factor. immunoprecipitates were washed twice in lysis buer. The immunoprecipitate/agarose complexes were recovered after each wash by a two minute centrifugation at 10 000 g at 48C.
In vitro kinase assay Acknowledgements The immunoprecipitates were washed once in kinase buer We are grateful to Jun Myoshi for kindly providing (50 mM HEPES, pH 7.5, 50 mM b-glycerophosphate, 3 mM dierent cot expression plasmids and Cot antibody. We
EGTA, 10% (v/v) glycerol, 2 mM DTT, 20 mM MgCl2, further thank Silvia PfraÈ nger for excellent photographic 200 mM ATP). Samples were resuspended in 30 mlofkinase reproduction, Viktor Wixler, Barbara Bauer and Manuela buer containing 5 mCi [g-32P]ATP (3000 Ci/mM,Amer- Schuler for technical assistance, and Carsten Hagemann sham) and 4 mg of recombinant K97M kinase dead MEK and Bruce Jordan for critical reading of the manuscript. Activation of MEK-1 and SEK-1 by Cot D Hagemann et al 1400 References
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