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Nuclear translocation of p42/p44 mitogen‐activated protein kinase is required for growth factor‐induced gene expression and cell cycle entry

Nuclear translocation of p42/p44 mitogen‐activated protein kinase is required for growth... The EMBO Journal Vol.18 No.3 pp.664–674, 1999 Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry response to nerve growth factor (NGF) (Traverse et al., Anne Brunet , Danie ` le Roux, 1992; Alessi et al., 1995) and long-term potentiation in Philippe Lenormand, Stephen Dowd , 2 3 presynaptic neurons in Aplysia in response to serotonin Stephen Keyse and Jacques Pouysse ´ gur (Martin et al., 1997). CNRS-UMR 6543 Centre de Biochimie, Universite´ de Nice, Parc Both p42 and p44MAPK are activated by dual phospho- Valrose, 06108 Nice, France and ICRF Molecular Pharmacology Unit, rylation on a threonine and a tyrosine residue, achieved Biomedical Reseach Centre, Level 5, Ninewells Hospital, Dundee by the dual-specificity kinase MKK1/2. Whereas MKK1/2 DD1 9SY, UK remain permanently in the cytoplasm, p42/p44MAPK Present address: M.E.Greenberg’s Laboratory, Children’s Hospital, are relocalized from the cytoplasm to the nucleus upon Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, stimulation (Chen et al., 1992; Gonzalez et al., 1993; USA Lenormand et al., 1993; Zheng and Guan, 1994). In Corresponding author fibroblasts, a correlation exists between the mitogenic e-mail: [email protected] potency of a stimulus and its ability to trigger MAPK translocation. This correlation is also found in PC12 cells Mitogen-activated protein kinase (MAPK) modules, (Traverse et al., 1992) and in Aplysia (Martin et al., 1997). composed of three protein kinases activated by success- Comparison of the kinetics of MAPK activation and ive phosphorylation, are involved in the signal trans- nuclear translocation suggests that it is the active form of duction of a wide range of extracellular agents. In MAPK that translocates into the nucleus (Lenormand mammalian cells, mitogenic stimulation triggers the et al., 1993; Khokhlatchev et al., 1998). translocation of p42/p44MAPK from the cytoplasm to In mammalian cells, inactivation of MAPK is achieved the nucleus, whereas the other protein kinases of the by a family of dual-specificity MAPK phosphatases which module remain cytosolic. Since MAPK has been shown target the two critical phosphorylation sites in the activa- to phosphorylate and activate nuclear targets, such as tion loop of MAPK (Keyse, 1998). Several members of the transcription factor Elk1, it has been proposed, this growing family of enzymes, including CL100 but not yet demonstrated, that MAPK nuclear translo- (MKP-1), MKP-2 and PAC-1, are tightly localized to the cation could represent a critical step in signal transduc- cell nucleus (Rohan et al., 1993; Brondello et al., 1995; tion. In this study, we sequestered p42/p44MAPK in Guan and Butch, 1995; King et al., 1995; Kwak and the cytoplasm by the expression of a catalytically Dixon, 1995). In addition, the genes encoding these inactive form of cytoplasmic MAP kinase phosphatase proteins are inducible by many of the stimuli which cause (MKP-3/Pyst-1). Sequestering MAPK in the cytoplasm MAPK activation, suggesting that they might be involved did not alter its activation or its ability to phosphorylate in feedback control of MAPK activity (Alessi et al., 1993; cytoplasmic substrates of MAPK (p90RSK1 or an Sun et al., 1993; Brondello et al., 1997). More recently, engineered cytoplasmic form of Elk1). In contrast, a novel subfamily of these proteins with atypical properties prevention of MAPK nuclear translocation strongly has been characterized, The first of these was MKP-3/ inhibited Elk1-dependent gene transcription and the ability of cells to reinitiate DNA replication in response Pyst-1 which, unlike MKP-1 and -2, is found predomi- to growth factors. Thus the relocalization of MAPK to nantly in the cytosol and is highly specific for p42/ the nucleus appears to be an important regulatory step p44MAPK (Groom et al., 1996; Muda et al., 1996). Thus, for mitogen-induced gene expression and cell cycle MAPK dephosphorylation and inactivation may take place re-entry. in both the nucleus and the cytoplasm, and individual Keywords: growth factors/MAP kinase/MAPK MKPs can be highly selective in targeting different MAPK phosphatase/nuclear translocation/substrates isoforms. p42 and p44MAPK are able to phosphorylate multiple substrates, found in various subcellular localizations: mem- brane-associated, such as the epidermal growth factor Introduction (EGF) receptor (Northwood et al., 1991), cytoplasmic, such as c-PLA2 (Lin et al., 1993), or nuclear, such as the Mitogen-activated protein kinase (MAPK) modules are Elk1/TCF (Gille et al., 1992; Janknecht et al., 1993; involved in the signal transduction of a wide variety of Marais et al., 1993) or c-Myc (Seth et al., 1992). Since signals in all eukaryotic organisms. In mammalian cells, many of the nuclear substrates of MAPK are transcription three well-characterized modules co-exist: p42/p44MAPK, factors, MAPK nuclear translocation is thought to represent p38MAPK and JNK cascades (for a review see Cano and Mahadevan, 1995). The p42/p44MAPK cascade plays a a crucial step in the modulation of gene expression. pivotal role in the re-entry of fibroblasts into the cell cycle However, it is possible that the cytoplasmic pool of active (Page`s et al., 1993), and in a variety of other processes, MAPK could phosphorylate transcription factors in the including differentiation of PC12 cells into neurites in cytoplasm when they are released from the synthetic 664 © European Molecular Biology Organization Role of p42/p44MAPK nuclear translocation machinery. More importantly, whereas a number of reports MKP-3 completely prevented MAPK activation upon have highlighted the role of MAPK activation in triggering growth factor stimulation, which is consistent with its different biological responses such as proliferation or phosphatase activity. In contrast, MKP-3 C/S did not block differentiation, it has never been addressed whether its the serum-stimulated activation of MAPK (Figure 1B). nuclear translocation and phosphorylation of nuclear tar- The fact that MKP-3 inactive mutant did not alter the gets was crucial for eliciting such responses or whether state of phosphorylation of MAPK was also confirmed by the phosphorylation of cytoplasmic substrates might be immunoblot (see below, Figure 3C, lower panel). There- sufficient. fore, the inactive form of MKP-3 specifically blocks To address the question of the significance of MAPK MAPK nuclear translocation, without affecting its nuclear translocation, we forced p42/p44MAPK cyto- phosphorylation by upstream MKK1/2. plasmic retention in fibroblasts by expressing an inactive form of the MKP-3 cytoplasmic phosphatase. These condi- The inactive form of MKP-3 affects the nuclear tions, which abolish MAPK nuclear translocation in translocation of p42/p44MAPK but not of response to serum, did not perturb MAPK activation p38MAPK or JNK or phosphorylation of cytoplasmic substrates. However, To determine whether the effect of the MKP-3 inactive enforced cytoplasmic retention of MAPK inhibited Elk1- mutant was specific towards p42/p44MAPK family mem- dependent gene expression and cell cycle re-entry in bers, we transfected MKP-3 C/S and analysed the localiz- response to growth factors. Our results support the notion ation of the endogenous forms of p42/p44MAPK, that regulating the accessibility of MAPK to the nucleus p38MAPK and JNK, using polyclonal antibodies directed is a key signalling event by which cells may control the to the different MAPKs. This experiment showed that intensity and temporal activation of genes during cell MKP-3 C/S retained p42/p44MAPK in the cytoplasm as growth and differentiation. demonstrated above, but did not affect the localization of the endogenous forms of p38MAPK or JNK (Figure 2A). To investigate this point further, we co-transfected Results MKP-3 C/S together with a hemagglutinin (HA)-tagged Expression of an inactive form of MKP-3 prevents form of p44MAPK, p38MAPK and JNK. When overex- MAPK nuclear translocation pressed, all three members of the MAPK family are To determine the importance of MAPK nuclear transloca- localized both in the cytoplasm and in the nucleus (Figure tion independently of its activation, we attempted to 2B). The expression of inactive MKP-3 retained retain MAPK in the cytoplasm without affecting its ectopically expressed HA-p44MAPK in the cytoplasm phosphorylation state. To this end, we used an inactive when MKP-3 was overexpressed relative to MAPK (with version of the MKP-3/Pyst1 phosphatase, because it dis- a 4:1 ratio) (Figure 2B). It has to be noted, however, that played the unique characteristics among the MKP family in cases where MAPK was overexpressed relative to of being localized in the cytoplasm and being able to form MKP-3, MAPK bypassed the cytoplasmic retention of a specific complex with p42/p44MAPK, to the exclusion MKP-3 and entered the nucleus (data not shown). In of the other MAPK family members (Groom et al., 1996; contrast to what was seen with p42/p44MAPK, the localiz- Muda et al., 1998). ation of neither JNK nor p38MAPK was affected by the We expressed in CCL39 fibroblasts Myc-tagged versions expression of inactive MKP-3 (Figure 2B). The MKP-3 of MKP-3, either wild-type (WT) or an inactive mutant inactive mutant, therefore, represents a useful and specific in which the crucial cysteine of the catalytic site has been tool to assay the role of p42/p44MAPK nuclear localization replaced by a serine (C/S), and determined the effect of in the regulation of downstream events. these constructs on the endogenous MAPK localization by double immunofluorescence. Both WT and inactive The inactive form of MKP-3 does not impair forms of MKP-3 were localized cytoplasmically and cytoplasmic substrate phosphorylation abrogated endogenous p42MAPK nuclear translocation If MKP-3 inactive mutant was only affecting the ability upon serum stimulation (Figure 1A). Quantification of MAPK to translocate into the nucleus, then we predicted revealed that 95% of the cells expressing MKP-3 (WT that it should not affect the phosphorylation of a cyto- or C/S) showed a retention of endogenous MAPK in the plasmic substrate of MAPK. cytoplasm. The cytoplasmic retention of MAPK also To address this issue, we first substituted part of the occurred in cells expressing much lower amounts of C-terminal cytoplasmic tail of an integral membrane MKP-3 (WT or C/S), as judged by immunofluorescence protein, the Na /H exchanger (NHE), with the region of (data not shown). Elk1 containing the four MAPK phosphorylation sites In agreement with Groom et al. (1996), experiments (Janknecht et al., 1993; Marais et al., 1993). As expected, using an antibody directed against the dual-phosphoryl- this cytoplasmically anchored form of Elk1 was localized ated, and thus active, form of MAPK show that WT at the plasma membrane (Figure 3A). Western blot analysis Fig. 1. Catalytically inactive mutant of MKP-3 phosphatase prevents endogenous p42MAPK nuclear translocation without affecting its activation. (A) CCL39 cells, transiently transfected with the expression vectors encoding Myc-tagged MKP-3 (WT or the C/S inactive mutant) were rendered quiescent and stimulated with 20% FCS for 3 h. The localization of MKP-3 was revealed by immunofluorescence using the anti-Myc antibody (Myc), whereas the localization of the endogenous p42MAPK was revealed with the MAPK-specific antibody (MAPK). Pictures shown are representative of at least three independent experiments conducted with different methods of fixation (see Materials and methods). (B) CCL39 cells, transiently transfected with the expression vectors encoding Myc-tagged MKP-3 (WT or the C/S inactive mutant), were rendered quiescent and stimulated with 20% FCS for 15 min. The localization of MKP-3 was revealed by immunofluorescence using the anti-Myc antibody (Myc), whereas phosphorylated MAPK was revealed by immunofluorescence using the phospho-MAPK antibody (P-MAPK). 665 A.Brunet et al. 666 Role of p42/p44MAPK nuclear translocation Fig. 2. Inactive form of MKP-3 does not alter the subcellular localization of either p38MAPK or JNK. (A) CCL39 cells were transfected with expression vectors encoding Myc-tagged MKP-3 C/S. The localization of the MKP-3 C/S was revealed by immunofluorescence using the anti-Myc antibody (Myc), whereas the localization of the endogenous members of the MAPK family was revealed by specific polyclonal antibodies (see Materials and methods). (B) CCL39 cells were co-transfected with expression vectors encoding MKP-3 C/S mutant and vectors encoding HA-p44MAPK, HA-p38MAPK and HA-JNK1 in a 4:1 ratio. The localization of MKP-3 was assessed with the anti-MKP-3 polyclonal antibody, whereas the localization of the different members of the MAPK family was revealed by the anti-HA antibody. 667 A.Brunet et al. Fig. 3. Inactive form of MKP-3 does not prevent the phosphorylation of a cytoplasmic form of Elk1. (A) CCL39 cells were transiently transfected with the construct encoding the fusion between the Na /H exchanger (NHE1) and Elk1 (NHE–Elk1). The subcellular localization of this construct was assayed by immunofluorescence, using an anti-Elk1 antibody. (B) A stable CCL39 cell line expressing both the Raf::ER chimera and the NHE–Elk1 fusion protein was rendered quiescent for 16 h and stimulated with increasing concentrations of estradiol for 30 min, or pre-treated with the MKK1 inhibitor PD 098059 for 30 min and then stimulated with 30 nM estradiol for 30 min. Membrane fractions were resolved on SDS–PAGE and blotted with an anti-Elk 1 antibody. (C) CCL39 cells expressing the Raf::ER chimeric construct were transiently co-transfected with empty vector (V), MKP-3 WT, MKP-3 C/S and NHE–Elk1. Cells resistant to the acid load selection were rendered quiescent and stimulated with 30 nM estradiol for 30 min. Membrane fractions were resolved on SDS–PAGE and blotted with an anti-Elk1 antibody (upper panel). Cytoplasmic fractions were resolved on SDS–PAGE and blotted with an anti-p42MAPK antibody (lower panel). The immunoblots shown are representative of two independent experiments. conducted with an anti-Elk1 antibody shows that in fibroblasts expressing the Raf::ER chimeric protein, estra- diol stimulation, which specifically activates the Raf– MAPK cascade, led to a retardation of the electrophoretic mobility of NHE–Elk1. This mobility shift is MAPK- dependent as it is abolished by the MKK1-specific inhibitor PD 098059 (Dudley et al., 1995) (Figure 3B). Altogether, these results indicate that we have engineered a cyto- plasmic form of Elk1 which, like WT Elk1, is dependent on MAPK activation for phosphorylation. We next asked whether the estradiol-stimulated phosphorylation of NHE–Elk1 was affected by the expres- sion of MKP-3. As expected, the WT form of MKP-3 was able to suppress the phosphorylation shift of NHE– Elk1 induced by estradiol (Figure 3C, upper panel, com- Fig. 4. Inactive form of MKP-3 does not prevent the phosphorylation pare lanes 2 and 4). In contrast, this mobility shift was of the cytoplasmic fraction of p90RSK1. 293 cells were transiently not affected by the inactive form of MKP-3 (Figure 3C, co-transfected with empty vector (V), or vectors encoding MKP-3 upper panel, compare lanes 2 and 3). (WT or the C/S inactive mutant) and the construct encoding HA- To extend this finding to other substrates of MAPK and p90RSK1. Cells were starved for 20 h (–) and stimulated for 10 min obtain more quantitative analysis on the phosphorylation with 20% FCS (). The cytoplasmic fraction of HA-p90RSK1 was immunoprecipitated with the anti-HA antibody, and its kinase activity of cytoplasmic substrate in cells expressing the inactive was assayed using CREB as a substrate in the presence of [γ- P]ATP form of MKP-3, we assayed the activity of the kinase (upper panel). The expression of HA-p90RSK1 was determined by p90RSK1, which is dependent upon its phosphorylation immunoblot using the anti-p90RSK1 antibody (middle panel). The by p42/p44MAPK (Grove et al., 1993). p90RSK1 has expression of MKP-3 was monitored by immunoblot using the anti- been shown to be cytoplasmic under resting conditions Myc antibody (lower panel). but can translocate to the nucleus upon growth factor stimulation (Chen et al., 1992). We therefore prepared cytoplasmic fractions of 293 cells transfected with Taken together, these experiments indicate that the MKP-3 and assayed the kinase activity of p90RSK1 in inactive form of MKP-3, although binding tightly to such fractions. The inactive MKP-3 C/S mutant, in contrast MAPK and preventing its nuclear translocation, did not to the WT form, did not alter p90RSK1 activation impair MAPK’s ability to phosphorylate cytoplasmic sub- (Figure 4). strates. 668 Role of p42/p44MAPK nuclear translocation the exogenous expression of high levels of p44MAPK (Figure 5B), but not by lower levels (data not shown). This is consistent with the fact that overexpression of MAPK bypasses the cytoplasmic trapping achieved by MKP-3 C/S. In contrast, the overexpression of neither p38MAPK nor JNK reversed the MKP-3 C/S negative effect on Gal4–Elk1 activity (Figure 5B). These results indicate that MAPK nuclear translocation is essential to maximally phosphorylate and activate one of its nuclear targets, Elk1. The inactive form of MKP-3 reduces the rate of S phase entry in response to mitogenic stimulation We then asked more generally whether the nuclear translo- cation of MAPK was necessary for the mitogenic response of the cells. To examine whether the prevention of MAPK nuclear translocation interferes with G /S phase progres- sion, we monitored DNA replication by bromodeoxyurid- ine (BrdU) incorporation. In contrast to cells expressing the PTP1C phosphatase, which apparently plays no role in S phase entry (Rivard et al., 1995), cells expressing WT MKP-3 were unable to enter S phase (Figure 6A and B). More importantly, cells expressing the inactive form of MKP-3 were also blocked in the G phase (Figure 6A and B). This suggested that the blockage of MAPK nuclear translocation prevented G /S phase progression. To exclude the possibility that MKP-3 could enter the nucleus transiently, as a shuttling molecule, we targeted MKP-3 C/S to the plasma membrane by generating a fusion protein with the sodium/proton exchanger (NHE– MKP-3 C/S). Ectopic expression of membrane-targeted MKP-3 C/S prevented MAPK nuclear translocation (data not shown) and also prevented DNA synthesis, whereas NHE alone did not (Figure 6A and B). Interestingly, similar results were obtained by expressing a version of NHE–MKP-3 in which we deleted the phosphatase cata- lytic domain, leaving intact the N-terminal MAPK-inter- Fig. 5. Inactive form of MKP-3 inhibits nuclear Elk1 activity. acting domain (Muda et al., 1998) (see Materials and (A) CCL39 cells were transiently transfected with the construct methods). This construct, when overexpressed, prevented encoding the Gal4–Elk1 fusion protein. The subcellular localization of this construct was assayed by immunofluorescence, using an anti-Elk1 MAPK nuclear translocation and G /S progression up to antibody. (B) CCL39 cells were transiently co-transfected with the 70% (data not shown). empty vector (pSG5), or vectors encoding MKP-3 (WT or the C/S As was the case for the luciferase activity (see Figure inactive mutant), Gal4–Elk1 fusion protein and the Gal4–luciferase 5B), the overexpression of p44MAPK reversed the reporter constructs. Vectors encoding HA-p44MAPK, HA-p38MAPK blockage of G /S phase progression due to the expression or HA-JNK were added at a 2:1 ratio compared with the MKP-3 1 vectors. Luciferase activity was assayed 2 days after transfection. The of MKP-3 inactive mutant but not that of the WT form values represent the mean  SEM of four independent experiments (Figure 6C). This reversion was specific to p44MAPK conducted in triplicate. and was not observed with either JNK or p38MAPK (Figure 6C). The inactive form of MKP-3 prevents nuclear Altogether, these experiments strongly suggest that Elk1-dependent transcription MAPK nuclear translocation is a crucial step in signal We asked whether the blockage of MAPK nuclear translo- transduction leading to gene expression and promoting cation affected the activation of MAPK-dependent nuclear cell cycle re-entry upon mitogenic stimulation. targets, such as transcription factors. To this end, we took advantage of the Gal4–Elk1 fusion protein which is Discussion localized predominantly in the nucleus (Figure 5A) and whose phosphorylation and subsequent activation by In this study, we have reported a means to specifically MAPK can be assayed using a Gal4–luciferase reporter prevent p42/p44MAPK nuclear translocation without gene. As expected, expression of MKP-3 WT which affecting its activation. Different methods can theoretically potently inactivates p42/p44MAPK (see Figure 1B) inhib- be used to achieve the blockage of MAPK nuclear translo- ited Gal4–Elk1 activation (Figure 5B). Interestingly, cation. The one we employed here was to create an expression of the inactive form of MKP-3 inhibited Gal4– artificial anchor for MAPK based on two criteria: a specific Elk1 activation by 60–70% (Figure 5B). The effect of interaction with MAPK and a cytoplasmic localization. MKP-3 C/S, but not that of MKP-3 WT, was reverted by Several proteins could possibly fulfil the criteria to create 669 A.Brunet et al. 670 Role of p42/p44MAPK nuclear translocation a cytoplasmic anchor for MAPK. For instance, the activator the endogenous form of MAPK (A.Brunet, unpublished of MAPK, MKK1, is a cytoplasmic protein that also binds observation). specifically to MAPK (Bardwell et al., 1996; Fukuda Therefore, preventing MAPK nuclear translocation by et al., 1997) and has therefore been proposed to play the anchoring it in the cytoplasm with an inactive phosphatase role of an MAPK anchor in vivo (Fukuda et al., 1997). represents an attractive method to assay the role of MAPK Moreover expression of MKK1 in Xenopus has been translocation. However, one potential drawback to this shown to impair MAPK nuclear translocation (Fukuda approach was that an efficient trapping of MAPK might et al., 1997). However, in our fibroblast CCL39 cell line, also prevent the accessibility to the upstream MKK1/2 or to the ability of MKK1 expression to prevent MAPK nuclear all the substrates. Our experiments, either using antibodies translocation was much weaker than that of inactive MKP- directed against the phosphorylated form of MAPK or 3. This can be explained by the fact that MKK1, when analysing the retardation in the electrophoretic mobility overexpressed, is less excluded from the nucleus than is of MAPK clearly showed that MAPK, in a complex with MKP-3, which is in agreement with the observation that MKP-3 C/S, was phosphorylated. This complexed MAPK MKK1 can enter the nucleus under certain circumstances was also active towards a substrate in vitro, as demon- (Jaaro et al., 1997). More importantly, MKK1 interacts strated with in vitro kinase assays (P.Lenormand, unpub- more weakly with MAPK than does MKP-3 (P.Lenormand, lished observation). As far as the accessibility to substrates unpublished observation). is concerned, we were able to show that when complexed Another method to prevent MAPK nuclear translocation to MKP-3, MAPK was still able to phosphorylate cyto- would be to affect its transport into the nucleus. The plasmic substrates in vivo. Therefore, the complex between mechanism by which MAPK is transported into the nucleus MKP-3 inactive mutant and MAPK appears to be tight is not yet known, but it has been hypothesized that enough to prevent MAPK nuclear translocation, but does transcription factors could co-transport with it, via a not affect MAPK general activation and substrate recogni- ‘piggy-back’ system. Recent experiments conducted with tion. The domains of MKP-3 involved in MAPK specificity Spc1, the p38MAPK homologue in fission yeast, show of recognition recently have been mapped in the N- that the deletion of the transcription factor Atf1, which is terminal region of the MKP-3 (Camps et al., 1998; Muda a major substrate of this kinase, prevented Spc1 relocaliz- et al., 1998). This domain alone, when targeted to the ation into the nucleus (Gaits et al., 1998). Previous work plasma membrane as a fusion with the NHE appears to from our laboratory has also supported the notion that be sufficient to prevent MAPK nuclear translocation. This nuclear proteins could play a role in MAPK nuclear result indicates that the cytoplasmic retention of MAPK translocation and retention (Lenormand et al., 1998). is mediated via the N-terminal MAPK-interacting site and However, interfering with transcription factors, which are is not as a result of ‘substrate trapping’ by the C/S mutant MAPK major targets, cannot be used to assay the role of of MKP-3. It would be interesting to determine the MAPK translocation in biological responses since these corresponding region of MAPK that interacts with MKP-3. responses may themselves require transcription. Since a mutant of MAPK resistant to MKP action previ- A third way to prevent MAPK nuclear translocation is ously had been mapped to the extreme C-terminal domain to act on MAPK itself. In a recent report, M.Cobb’s group of the MAPK (Bott et al., 1994; Camps et al., 1998), this showed that MAPK could form dimers when one of the region could be the one interacting with MKP-3. The fact monomers is phosphorylated and that this dimerization that the domains involved in the recognition of substrates could lead to MAPK nuclear translocation (Khokhlatchev were mapped in a more upstream domain of MAPK et al., 1998). Mutants of the C-terminal region of MAPK (Kallunki et al., 1994; Brunet and Pouysse´gur, 1996) which are unable to dimerize remain cytoplasmic even reinforces the observation that MAPK accessibility to upon growth factor stimulation (Khokhlatchev et al., substrate would not be impaired by its interaction with 1998). Since those mutants still retain their kinase activity, MKP-3. they could be used to assay the role of MAPK nuclear Using MKP-3 inactive mutant as a tool, we were able first translocation. Using a similar approach, we fused an NES to show that the blockage of MAPK nuclear translocation to the N- or C-terminal ends of p44MAPK in order to impaired MAPK-dependent activation of the transcription enforce its nuclear exit. However, these latter approaches factor Elk1. Although this result was expected, it had not rely both on the fact that MAPK dimer formation is yet been formally excluded that MAPK nuclear substrates efficient in vivo and that the introduction of a mutant form could have been phosphorylated in the cytoplasm, where of MAPK will also be able to prevent the endogenous they undergo obligatory transit during their biosynthesis. In form of MAPK from translocating into the nucleus, in a this alternative model, the relay between the cytoplasm and dominant-negative fashion. Preliminary results obtained the nucleus would have been established by transcription with the NES–MAPK indicate that these forms are not factors, as is the case for the JAK–STAT or the NF-κB able to function efficiently as dominant-negatives with pathways (Karin and Hunter, 1995). Since MAPK nuclear Fig. 6. Inactive MKP-3 mutant prevents S phase entry upon mitogenic stimulation. (A) CCL39 cells, either not transfected or transiently transfected with the expression vectors encoding PTP1C, MKP-3 (WT or C/S mutant), NHE or an NHE–MKP-3 C/S fusion construct were rendered quiescent for 24 h and stimulated for 24 h with 20% FCS, the BrdU being added for the last 4 h. Cells expressing the different constructs were revealed by immunofluorescence using polyclonal primary antibodies directed against each protein (left panel). Reinitiation of DNA synthesis was followed by BrdU incorporation, detected by an anti-BrdU monoclonal antibody (middle panel). The DNA was stained using DAPI (right panel). (B) Quanti- fication of the experiment presented in (A). Experiments were performed in duplicate, and 100 cells were counted for each condition. For PTP1C, MKP-3 WT and C/S, the values represent the mean and SEM of three independent experiments. (C) Expression vectors encoding HA-p44MAPK, HA-p38MAPK or HA-JNK were transfected in a 2:1 ratio compared with MKP-3. Experiments were conducted in the same way as in (A). Experiments were perfomed in duplicate and 100 cells were counted for each condition. 671 A.Brunet et al. rabbit antibodies were purchased from Sigma. Texas Red X-conjugated translocation is required for Elk1-dependent activation, our goat anti-mouse or anti-rabbit antibodies were from Molecular Probe. results suggest that the phosphorylation of Elk1 by MAPK The BrdU proliferation kit was purchased from Amersham. The MKK1 occurs in the nucleus. Moreover, the blockage of MAPK inhibitor PD 098059 was obtained from New England Biolabs. nuclear translocation inhibited the activity of the total c-fos Constructs promoter, confirming that MAPK entry into the nucleus The expression vectors encoding the Myc-tagged phosphatase Pyst1/MKP- is crucial for immediate early gene expression (A.Brunet, 3, either WT or an inactive mutant in which Cys293 of the catalytic site unpublished observation). Therefore, in the MAPK signal- has been replaced by a serine (C/S), were described previously (Groom ling pathway, the relay between the cytoplasm and the nuc- et al., 1996). The expression vectors encoding HA-tagged forms of leus appears to be constituted by MAPK itself. p44MAPK, p38MAPK (Brunet and Pouysse´gur, 1996), JNK1 (De´rijard et al., 1994) and p90RSK1 (Grove et al., 1993), the PTP1C phosphatase The approach of blocking the nuclear relocalization of (Rivard et al., 1995) and the human NHE1 (Sardet et al., 1989) have all MAPK could also be used to assay in which subcellular been described. The expression vectors encoding the Gal4–Elk1 fusion compartment different MAPK substrates are phosphoryl- protein and the Gal4–luciferase reporter gene have been described previ- ated. Another question that remains to be answered in terms ously (Janknecht et al., 1994) and were kindly provided by Dr K.Kelly. The vector encoding human Elk1 was kindly provided by Dr R.A.Hipskind. of compartmentalization is where the dephosphorylation The construct encoding a fusion protein between the NHE1 and the C- and consecutive inactivation of MAPK by the MKPs occurs terminal domain of Elk1 was generated by PCR, by inserting an ApaI in vivo. Observations made in Aplysia (Martin et al., 1997) restriction site at the level of amino acid 317 of Elk1 in-frame with the and in fibroblasts (P.Lenormand, in preparation) show that ApaI restriction site corresponding to amino acid 698 of NHE1. The after long-term stimulation, active MAPK is only found in construct encoding a fusion protein between NHE1 and MKP-3 C/S mutant was generated by PCR, by inserting an ApaI site at the level of the first the cytoplasm. Therefore, one important role for MAPK amino acid of MKP-3 C/S in-frame with the ApaI site corresponding to nuclear translocation could be to enter in contact with nuc- amino acid 698 of NHE1. The construct encoding a fusion protein between lear phosphatases (MKP-1 and -2 for instance) in order NHE1 and the N-terminal domain of MKP-3 was generated by PCR, using to shut down the signal. Endogenous MKP-3 could also the EcoRI site of MKP-3 and deleting the region encoding amino acids 230–359 of MKP-3, so that the last amino acid and the Myc epitope tag participate in signal extinction by dephosphorylating the were in-frame with the N-terminal domain of MKP-3. The constructs were cytoplasmic pool of MAPK. verified by sequencing. Finally, as far as biological responses are involved, our results show that MAPK nuclear translocation appears to Cell culture be required for progression into S phase in response to The Chinese hamster lung fibroblast cell line CCL39 (ATCC) or CCL39 expressing the chimeric Raf::ER chimeric construct (Lenormand et al., mitogenic stimulation. It would be interesting to test other 1996) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) biological responses that have been shown to be under the (Gibco-BRL) supplemented with 7.5% fetal calf serum (FCS; Biowhit- control of MAPK, such as differentiation of PC12 cells taker) and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) at (Traverse et al., 1992) or thymocytes (Alberola-Ila et al., 37°C in an atmosphere of 95% air, 5% CO . 1995; Crompton et al., 1996), survival of PC12 cells (Xia Immunofluorescence et al., 1995) or long-term potentiation in Aplysia (Martin CCL39 cells were plated onto glass coverslips at a density of 10 cells/ et al., 1997). Since most of them are dependent upon gene 35 mm dish. They were transfected by the calcium phosphate technique expression and protein synthesis, it seems likely that they with 7.5 μg of the different constructs. The day after transfection, cells will also be affected by MAPK retention in the cytoplasm. were rendered quiescent by incubation in serum-free medium for 16 h, and stimulated with various agonists. They were then fixed for 15 min at However, other MAPK-controlled responses, such as the –20°C with methanol/acetone (70:30 v/v) or methanol/formaldehyde (99:1 initiation of protein synthesis (Pause et al., 1994) or the v/v) or for 15 min in 10% formaldehyde at room temperature, followed by suppression of integrin activation (Hughes et al., 1997) may a methanol permeabilization for 10 min at –20°C for phospho-MAPK be independent of MAPK nuclear translocation. antibody. Coverslips were washed with phosphate-buffered saline (PBS) and the non-specific sites were blocked by incubation with PBS  3% In conclusion, our study provides a way to specifically bovine serum albumin (BSA). Coverslips were incubated with the first block MAPK nuclear translocation without affecting its antibody diluted in PBS/BSA (anti-HA, 1/500; anti-Myc, 1/100; anti- activation. In addition to demonstrating the importance of MAPK, 1/2000; anti-phosphoMAPK, 1/150; anti-p38MAPK, 1/300; anti- MAPK nuclear translocation for cellular processes such as JNK, 1/300; anti-Elk1, 1/500; anti-MKP-3, 1/100) for 1 h, then washed mitogen-induced gene expression and cell cycle progres- five times with PBS. Cells were incubated with the second antibody (goat anti-mouse FITC-conjugated, 1/100; goat anti-rabbit Texas Red- sion, this system should be useful for specifically blocking conjugated, 1/500) diluted in PBS/BSA for 1 h. After extensive washes in subsets of MAPK responses, an effect which is not possible PBS and in distilled water, coverslips were mounted in Mowiol (Calbi- when one interferes more generally with MAPK activation. ochem) and examined under epifluorescent illumination. BrdU incorporation CCL39 cells were transfected with the relevant constructs as described Materials and methods above. The day after transfection, cells were starved for 24 h and stimulated Material for 24 h with 20% FCS (Biowhittaker), with a pulse of BrdU (1/500) Monoclonal antibodies directed against the HA and Myc epitopes were for the last 4 h. Cells were fixed for 15 min at –20°C with methanol/ purchased respectively from Boehringer Mannheim and Santa Cruz. We formaldehyde (99:1 v/v) and incubated with primary antibody (anti-MKP- used commercially available polyclonal antibodies directed against 3, 1/100; anti-PTP1C; 1/250; anti-NHE, 1/500) prior to incubation with p42MAP kinase (Upstate Biotechnology), the dual-phosphorylated the secondary antibody (FITC anti-rabbit: 1/100) as described above. The p42MAPK (Promega), p38MAPK and JNK1/2 (Sigma), and p90RSK1 cells were then fixed in 3.7% formaldehyde for 10 min at room temperature, (Santa Cruz). Polyclonal antibodies directed against MKP-3/Pyst1 were rinsed in PBS and the chromatin was rendered accessible by a 10 min generated against the last 20 amino acids of the human isoform (Groom treatment with HCl (2 M). Cells were washed extensively with PBS and et al., 1996). The polyclonal antibodies against the PTP1C phosphatase blocked in PBS  10% FCS  0.1% Tween-20. The coverslips were were kindly provided by Dr E.R.Stanley (Rivard et al., 1995). The poly- incubated with the anti-BrdU monoclonal antibody (1/2) for 1 h, then clonal antibodies (RPc28) directed against the C-terminal region of the washed five times with PBS  0.1% Tween-20 (PBST). The secondary human NHE1 isoform were described previously (Sardet et al., 1990). The antibody (Texas Red anti-mouse, 1/500) was incubated for 45 min. DAPI purified CREB protein was kindly provided by Dr A.J.Shaywitz. (4,6 diamidine dihydrochloride; Boehringer Mannheim) was added for Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or anti- the last 15 min at a final concentration of 0.2 μg/ml. Cells were washed 672 Role of p42/p44MAPK nuclear translocation extensively in PBST then in distilled water and mounted in Mowiol (Cal- Alessi,D.R., Smythe,C. and Keyse,S.M. (1993) The human CL100 gene biochem). encodes a tyr/thr-protein phosphatase which potently and specifically inactivates MAP kinase and suppresses its activation by oncogenic ras Luciferase assays in Xenopus oocyte extracts. Oncogene, 8, 2015–2020. CCL39 cells were seeded in 24-well plates, at a density of 10 cells/well, Alessi,D.R., Cuenda,A., Cohen,P., Dudley,D.T. and Saltiel,A.R. (1995) and were co-transfected with 0.25 μg of the 5 Gal4–luciferase reporter PD 098059 is a specific inhibitor of the activation of mitogen-activated gene, 0.5 μg of the Gal4–Elk1 fusion construct, 0.5 μg of the the constructs protein kinase kinase in vitro and in vivo. J. Biol. Chem., 270, 27489– corresponding to MKP-3 and 1 μg of the constructs corresponding to the different MAPK family members. Two days after transfection, cells were Bardwell,L., Cook,J.G., Chang,E.C., Cairns,B.R. and Thorner,J. (1996) lysed in 100 μl of lysis buffer and the luciferase activity of one-tenth of Signaling in the yeast pheromone response pathway: specific and high- the samples was assayed according to the Promega protocol. affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7. Mol. Cell. Biol., Western blotting 16, 3637–3650. 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Oncogene, 1085, 1895–1904. Membrane fractions were obtained by incubating the cells in a hypotonic Brondello,J.-M., Brunet,A., Pouysse´gur,J. and McKenzie,F.R. (1997) The buffer[10 mMTris–HCl pH8.0,1 mMEDTA,1 mMphenylmethylsulfonyl dual specificity mitogen-activated protein kinase phosphatase-1 and fluoride (PMSF), 1 mM phenanthroline, 1 mM iodoacetamide] for 10 min -2 are induced by the p42/p44MAPK cascade. J. Biol. Chem., 272, at 4°C. Cells were then scrapped and the membranes were pelleted by 1368–1376. centrifugation at 40 000 r.p.m. for 20 min at 4°C and directly resuspended Brunet,A. and Pouysse´gur,J. (1996) Identification of MAP kinase domains in 1 Laemmli sample buffer. Proteins were resolved on SDS–PAGE by redirecting stress signals into growth factor responses. Science, 272, (7.5%; 19:1 acrylamide/bisacrylamide) and transferred onto PVDF mem- 1652–1655. branes (Immobilon). The membranes were incubated with the anti-Elk1 Camps,M., Nichols,A., Gillieron,C., Antonsson,B., Muda,M., Chabert,C., antibody (1/1000) overnight at 4°C. Signal was detected using horseradish Boschert,U. and Arkinstall,S. (1998) Catalytic activation of the peroxidase (HRP)-conjugated anti-rabbit antibodies and the enhanced phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. chemiluminescencemethod. Cytoplasmicfractions wereresolvedon SDS– Science, 280, 1262–1265. PAGE (12.5%; 30:0.2 acrylamide/bisacrylamide) and immunoblotted with Cano,E. and Mahadevan,L.C. (1995) Parallel signal processing among the anti-p42MAPK antibody. mammalian MAPKs. Trends Biochem. Sci., 20, 117–122. Chen,R.H., Sarnecki,C. and Blenis,J. (1992) Nuclear localization and Kinase assay regulation of ERK- and RSK-encoded protein kinases. Mol. Cell. Biol., 293 cells were seeded in 35 mm dishes at a density of 750 000 cells per 12, 915–927. dish. They were transfected by the calcium phosphate technique with 5 μg Crompton,T., Gilmour,K.C. and Owen,M.J. (1996) The MAP kinase of the vector encoding HA-p90RSK1 and 1 μg of the vector encoding the pathway controls differentiation from double-negative to double- relevant constructs. positive thymocyte. Cell, 86, 243–251. At 24 h after transfection, cells were starved in serum-free medium for De´rijard,B., Hibi,M., Wu,I.-H., Barrett,T., Su,B., Deng,T., Karin,M. and 20 h, then stimulated with 20% FCS for 10 min. Cytoplasmic fractions Davis,R.J. (1994) JNK1: a protein kinase stimulated by UV light and were obtained by lysing the cells in a hypotonic buffer [5 mM Tris–HCl, Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell, pH 7.4, 1 mM MgCl 2 mM EGTA, 2 mM dithiothreitol (DTT), 5 mM 2, 76, 1025–1037. β-glycerophosphate, 1 mM PMSF, 1 mM aprotinin]. Extracts were Dounce Dudley,D.T., Pang,L., Decker,S.J., Bridges,A.J. and Saltiel,A.R. (1995) A homogenized 40 times and centrifuged at 10 000 r.p.m. for 5 min. The synthetic inhibitor of the mitogen-activated protein kinase cascade. supernatent was centrifuged at 15 000 r.p.m. for 30 min. Cytoplasmic Proc. Natl Acad. Sci. USA, 92, 7686–7689. extracts were immunoprecipitated with the anti-HA antibody coupled to Fukuda,M., Gotoh,Y. and Nishida,E. (1997) Interaction of MAP kinase protein A–Sepharose beads for 2 h. The immunoprecipitate was washed with MAP kinase kinase: its possible role in the control of twice in buffer A (10 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, nucleocytoplasmic transport of MAP kinase. EMBO J., 16, 1901–1908. 1% NP-40, 2 mM DTT, 40 mM β-glycerophosphate, 50 mM NaF, 1 mM Gaits,F., Degols,G., Shiozaki,K. and Russell,P. 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(1984) A specific mutation abolishing Na /H antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc. Natl Acad. Sci. USA, 81, 4833–4837. Rivard,N., McKenzie,F.M., Brondello,J.M. and Pouysse´gur,J. (1995) The phosphotyrosine phosphatase PTP1D, but not PTP1C, is an essential mediator of fibroblast proliferation induced by tyrosine kinase and G protein-coupled receptors. J. Biol. Chem., 270, 11017–11024. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Nuclear translocation of p42/p44 mitogen‐activated protein kinase is required for growth factor‐induced gene expression and cell cycle entry

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Copyright © European Molecular Biology Organization 1999
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10.1093/emboj/18.3.664
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Abstract

The EMBO Journal Vol.18 No.3 pp.664–674, 1999 Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry response to nerve growth factor (NGF) (Traverse et al., Anne Brunet , Danie ` le Roux, 1992; Alessi et al., 1995) and long-term potentiation in Philippe Lenormand, Stephen Dowd , 2 3 presynaptic neurons in Aplysia in response to serotonin Stephen Keyse and Jacques Pouysse ´ gur (Martin et al., 1997). CNRS-UMR 6543 Centre de Biochimie, Universite´ de Nice, Parc Both p42 and p44MAPK are activated by dual phospho- Valrose, 06108 Nice, France and ICRF Molecular Pharmacology Unit, rylation on a threonine and a tyrosine residue, achieved Biomedical Reseach Centre, Level 5, Ninewells Hospital, Dundee by the dual-specificity kinase MKK1/2. Whereas MKK1/2 DD1 9SY, UK remain permanently in the cytoplasm, p42/p44MAPK Present address: M.E.Greenberg’s Laboratory, Children’s Hospital, are relocalized from the cytoplasm to the nucleus upon Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, stimulation (Chen et al., 1992; Gonzalez et al., 1993; USA Lenormand et al., 1993; Zheng and Guan, 1994). In Corresponding author fibroblasts, a correlation exists between the mitogenic e-mail: [email protected] potency of a stimulus and its ability to trigger MAPK translocation. This correlation is also found in PC12 cells Mitogen-activated protein kinase (MAPK) modules, (Traverse et al., 1992) and in Aplysia (Martin et al., 1997). composed of three protein kinases activated by success- Comparison of the kinetics of MAPK activation and ive phosphorylation, are involved in the signal trans- nuclear translocation suggests that it is the active form of duction of a wide range of extracellular agents. In MAPK that translocates into the nucleus (Lenormand mammalian cells, mitogenic stimulation triggers the et al., 1993; Khokhlatchev et al., 1998). translocation of p42/p44MAPK from the cytoplasm to In mammalian cells, inactivation of MAPK is achieved the nucleus, whereas the other protein kinases of the by a family of dual-specificity MAPK phosphatases which module remain cytosolic. Since MAPK has been shown target the two critical phosphorylation sites in the activa- to phosphorylate and activate nuclear targets, such as tion loop of MAPK (Keyse, 1998). Several members of the transcription factor Elk1, it has been proposed, this growing family of enzymes, including CL100 but not yet demonstrated, that MAPK nuclear translo- (MKP-1), MKP-2 and PAC-1, are tightly localized to the cation could represent a critical step in signal transduc- cell nucleus (Rohan et al., 1993; Brondello et al., 1995; tion. In this study, we sequestered p42/p44MAPK in Guan and Butch, 1995; King et al., 1995; Kwak and the cytoplasm by the expression of a catalytically Dixon, 1995). In addition, the genes encoding these inactive form of cytoplasmic MAP kinase phosphatase proteins are inducible by many of the stimuli which cause (MKP-3/Pyst-1). Sequestering MAPK in the cytoplasm MAPK activation, suggesting that they might be involved did not alter its activation or its ability to phosphorylate in feedback control of MAPK activity (Alessi et al., 1993; cytoplasmic substrates of MAPK (p90RSK1 or an Sun et al., 1993; Brondello et al., 1997). More recently, engineered cytoplasmic form of Elk1). In contrast, a novel subfamily of these proteins with atypical properties prevention of MAPK nuclear translocation strongly has been characterized, The first of these was MKP-3/ inhibited Elk1-dependent gene transcription and the ability of cells to reinitiate DNA replication in response Pyst-1 which, unlike MKP-1 and -2, is found predomi- to growth factors. Thus the relocalization of MAPK to nantly in the cytosol and is highly specific for p42/ the nucleus appears to be an important regulatory step p44MAPK (Groom et al., 1996; Muda et al., 1996). Thus, for mitogen-induced gene expression and cell cycle MAPK dephosphorylation and inactivation may take place re-entry. in both the nucleus and the cytoplasm, and individual Keywords: growth factors/MAP kinase/MAPK MKPs can be highly selective in targeting different MAPK phosphatase/nuclear translocation/substrates isoforms. p42 and p44MAPK are able to phosphorylate multiple substrates, found in various subcellular localizations: mem- brane-associated, such as the epidermal growth factor Introduction (EGF) receptor (Northwood et al., 1991), cytoplasmic, such as c-PLA2 (Lin et al., 1993), or nuclear, such as the Mitogen-activated protein kinase (MAPK) modules are Elk1/TCF (Gille et al., 1992; Janknecht et al., 1993; involved in the signal transduction of a wide variety of Marais et al., 1993) or c-Myc (Seth et al., 1992). Since signals in all eukaryotic organisms. In mammalian cells, many of the nuclear substrates of MAPK are transcription three well-characterized modules co-exist: p42/p44MAPK, factors, MAPK nuclear translocation is thought to represent p38MAPK and JNK cascades (for a review see Cano and Mahadevan, 1995). The p42/p44MAPK cascade plays a a crucial step in the modulation of gene expression. pivotal role in the re-entry of fibroblasts into the cell cycle However, it is possible that the cytoplasmic pool of active (Page`s et al., 1993), and in a variety of other processes, MAPK could phosphorylate transcription factors in the including differentiation of PC12 cells into neurites in cytoplasm when they are released from the synthetic 664 © European Molecular Biology Organization Role of p42/p44MAPK nuclear translocation machinery. More importantly, whereas a number of reports MKP-3 completely prevented MAPK activation upon have highlighted the role of MAPK activation in triggering growth factor stimulation, which is consistent with its different biological responses such as proliferation or phosphatase activity. In contrast, MKP-3 C/S did not block differentiation, it has never been addressed whether its the serum-stimulated activation of MAPK (Figure 1B). nuclear translocation and phosphorylation of nuclear tar- The fact that MKP-3 inactive mutant did not alter the gets was crucial for eliciting such responses or whether state of phosphorylation of MAPK was also confirmed by the phosphorylation of cytoplasmic substrates might be immunoblot (see below, Figure 3C, lower panel). There- sufficient. fore, the inactive form of MKP-3 specifically blocks To address the question of the significance of MAPK MAPK nuclear translocation, without affecting its nuclear translocation, we forced p42/p44MAPK cyto- phosphorylation by upstream MKK1/2. plasmic retention in fibroblasts by expressing an inactive form of the MKP-3 cytoplasmic phosphatase. These condi- The inactive form of MKP-3 affects the nuclear tions, which abolish MAPK nuclear translocation in translocation of p42/p44MAPK but not of response to serum, did not perturb MAPK activation p38MAPK or JNK or phosphorylation of cytoplasmic substrates. However, To determine whether the effect of the MKP-3 inactive enforced cytoplasmic retention of MAPK inhibited Elk1- mutant was specific towards p42/p44MAPK family mem- dependent gene expression and cell cycle re-entry in bers, we transfected MKP-3 C/S and analysed the localiz- response to growth factors. Our results support the notion ation of the endogenous forms of p42/p44MAPK, that regulating the accessibility of MAPK to the nucleus p38MAPK and JNK, using polyclonal antibodies directed is a key signalling event by which cells may control the to the different MAPKs. This experiment showed that intensity and temporal activation of genes during cell MKP-3 C/S retained p42/p44MAPK in the cytoplasm as growth and differentiation. demonstrated above, but did not affect the localization of the endogenous forms of p38MAPK or JNK (Figure 2A). To investigate this point further, we co-transfected Results MKP-3 C/S together with a hemagglutinin (HA)-tagged Expression of an inactive form of MKP-3 prevents form of p44MAPK, p38MAPK and JNK. When overex- MAPK nuclear translocation pressed, all three members of the MAPK family are To determine the importance of MAPK nuclear transloca- localized both in the cytoplasm and in the nucleus (Figure tion independently of its activation, we attempted to 2B). The expression of inactive MKP-3 retained retain MAPK in the cytoplasm without affecting its ectopically expressed HA-p44MAPK in the cytoplasm phosphorylation state. To this end, we used an inactive when MKP-3 was overexpressed relative to MAPK (with version of the MKP-3/Pyst1 phosphatase, because it dis- a 4:1 ratio) (Figure 2B). It has to be noted, however, that played the unique characteristics among the MKP family in cases where MAPK was overexpressed relative to of being localized in the cytoplasm and being able to form MKP-3, MAPK bypassed the cytoplasmic retention of a specific complex with p42/p44MAPK, to the exclusion MKP-3 and entered the nucleus (data not shown). In of the other MAPK family members (Groom et al., 1996; contrast to what was seen with p42/p44MAPK, the localiz- Muda et al., 1998). ation of neither JNK nor p38MAPK was affected by the We expressed in CCL39 fibroblasts Myc-tagged versions expression of inactive MKP-3 (Figure 2B). The MKP-3 of MKP-3, either wild-type (WT) or an inactive mutant inactive mutant, therefore, represents a useful and specific in which the crucial cysteine of the catalytic site has been tool to assay the role of p42/p44MAPK nuclear localization replaced by a serine (C/S), and determined the effect of in the regulation of downstream events. these constructs on the endogenous MAPK localization by double immunofluorescence. Both WT and inactive The inactive form of MKP-3 does not impair forms of MKP-3 were localized cytoplasmically and cytoplasmic substrate phosphorylation abrogated endogenous p42MAPK nuclear translocation If MKP-3 inactive mutant was only affecting the ability upon serum stimulation (Figure 1A). Quantification of MAPK to translocate into the nucleus, then we predicted revealed that 95% of the cells expressing MKP-3 (WT that it should not affect the phosphorylation of a cyto- or C/S) showed a retention of endogenous MAPK in the plasmic substrate of MAPK. cytoplasm. The cytoplasmic retention of MAPK also To address this issue, we first substituted part of the occurred in cells expressing much lower amounts of C-terminal cytoplasmic tail of an integral membrane MKP-3 (WT or C/S), as judged by immunofluorescence protein, the Na /H exchanger (NHE), with the region of (data not shown). Elk1 containing the four MAPK phosphorylation sites In agreement with Groom et al. (1996), experiments (Janknecht et al., 1993; Marais et al., 1993). As expected, using an antibody directed against the dual-phosphoryl- this cytoplasmically anchored form of Elk1 was localized ated, and thus active, form of MAPK show that WT at the plasma membrane (Figure 3A). Western blot analysis Fig. 1. Catalytically inactive mutant of MKP-3 phosphatase prevents endogenous p42MAPK nuclear translocation without affecting its activation. (A) CCL39 cells, transiently transfected with the expression vectors encoding Myc-tagged MKP-3 (WT or the C/S inactive mutant) were rendered quiescent and stimulated with 20% FCS for 3 h. The localization of MKP-3 was revealed by immunofluorescence using the anti-Myc antibody (Myc), whereas the localization of the endogenous p42MAPK was revealed with the MAPK-specific antibody (MAPK). Pictures shown are representative of at least three independent experiments conducted with different methods of fixation (see Materials and methods). (B) CCL39 cells, transiently transfected with the expression vectors encoding Myc-tagged MKP-3 (WT or the C/S inactive mutant), were rendered quiescent and stimulated with 20% FCS for 15 min. The localization of MKP-3 was revealed by immunofluorescence using the anti-Myc antibody (Myc), whereas phosphorylated MAPK was revealed by immunofluorescence using the phospho-MAPK antibody (P-MAPK). 665 A.Brunet et al. 666 Role of p42/p44MAPK nuclear translocation Fig. 2. Inactive form of MKP-3 does not alter the subcellular localization of either p38MAPK or JNK. (A) CCL39 cells were transfected with expression vectors encoding Myc-tagged MKP-3 C/S. The localization of the MKP-3 C/S was revealed by immunofluorescence using the anti-Myc antibody (Myc), whereas the localization of the endogenous members of the MAPK family was revealed by specific polyclonal antibodies (see Materials and methods). (B) CCL39 cells were co-transfected with expression vectors encoding MKP-3 C/S mutant and vectors encoding HA-p44MAPK, HA-p38MAPK and HA-JNK1 in a 4:1 ratio. The localization of MKP-3 was assessed with the anti-MKP-3 polyclonal antibody, whereas the localization of the different members of the MAPK family was revealed by the anti-HA antibody. 667 A.Brunet et al. Fig. 3. Inactive form of MKP-3 does not prevent the phosphorylation of a cytoplasmic form of Elk1. (A) CCL39 cells were transiently transfected with the construct encoding the fusion between the Na /H exchanger (NHE1) and Elk1 (NHE–Elk1). The subcellular localization of this construct was assayed by immunofluorescence, using an anti-Elk1 antibody. (B) A stable CCL39 cell line expressing both the Raf::ER chimera and the NHE–Elk1 fusion protein was rendered quiescent for 16 h and stimulated with increasing concentrations of estradiol for 30 min, or pre-treated with the MKK1 inhibitor PD 098059 for 30 min and then stimulated with 30 nM estradiol for 30 min. Membrane fractions were resolved on SDS–PAGE and blotted with an anti-Elk 1 antibody. (C) CCL39 cells expressing the Raf::ER chimeric construct were transiently co-transfected with empty vector (V), MKP-3 WT, MKP-3 C/S and NHE–Elk1. Cells resistant to the acid load selection were rendered quiescent and stimulated with 30 nM estradiol for 30 min. Membrane fractions were resolved on SDS–PAGE and blotted with an anti-Elk1 antibody (upper panel). Cytoplasmic fractions were resolved on SDS–PAGE and blotted with an anti-p42MAPK antibody (lower panel). The immunoblots shown are representative of two independent experiments. conducted with an anti-Elk1 antibody shows that in fibroblasts expressing the Raf::ER chimeric protein, estra- diol stimulation, which specifically activates the Raf– MAPK cascade, led to a retardation of the electrophoretic mobility of NHE–Elk1. This mobility shift is MAPK- dependent as it is abolished by the MKK1-specific inhibitor PD 098059 (Dudley et al., 1995) (Figure 3B). Altogether, these results indicate that we have engineered a cyto- plasmic form of Elk1 which, like WT Elk1, is dependent on MAPK activation for phosphorylation. We next asked whether the estradiol-stimulated phosphorylation of NHE–Elk1 was affected by the expres- sion of MKP-3. As expected, the WT form of MKP-3 was able to suppress the phosphorylation shift of NHE– Elk1 induced by estradiol (Figure 3C, upper panel, com- Fig. 4. Inactive form of MKP-3 does not prevent the phosphorylation pare lanes 2 and 4). In contrast, this mobility shift was of the cytoplasmic fraction of p90RSK1. 293 cells were transiently not affected by the inactive form of MKP-3 (Figure 3C, co-transfected with empty vector (V), or vectors encoding MKP-3 upper panel, compare lanes 2 and 3). (WT or the C/S inactive mutant) and the construct encoding HA- To extend this finding to other substrates of MAPK and p90RSK1. Cells were starved for 20 h (–) and stimulated for 10 min obtain more quantitative analysis on the phosphorylation with 20% FCS (). The cytoplasmic fraction of HA-p90RSK1 was immunoprecipitated with the anti-HA antibody, and its kinase activity of cytoplasmic substrate in cells expressing the inactive was assayed using CREB as a substrate in the presence of [γ- P]ATP form of MKP-3, we assayed the activity of the kinase (upper panel). The expression of HA-p90RSK1 was determined by p90RSK1, which is dependent upon its phosphorylation immunoblot using the anti-p90RSK1 antibody (middle panel). The by p42/p44MAPK (Grove et al., 1993). p90RSK1 has expression of MKP-3 was monitored by immunoblot using the anti- been shown to be cytoplasmic under resting conditions Myc antibody (lower panel). but can translocate to the nucleus upon growth factor stimulation (Chen et al., 1992). We therefore prepared cytoplasmic fractions of 293 cells transfected with Taken together, these experiments indicate that the MKP-3 and assayed the kinase activity of p90RSK1 in inactive form of MKP-3, although binding tightly to such fractions. The inactive MKP-3 C/S mutant, in contrast MAPK and preventing its nuclear translocation, did not to the WT form, did not alter p90RSK1 activation impair MAPK’s ability to phosphorylate cytoplasmic sub- (Figure 4). strates. 668 Role of p42/p44MAPK nuclear translocation the exogenous expression of high levels of p44MAPK (Figure 5B), but not by lower levels (data not shown). This is consistent with the fact that overexpression of MAPK bypasses the cytoplasmic trapping achieved by MKP-3 C/S. In contrast, the overexpression of neither p38MAPK nor JNK reversed the MKP-3 C/S negative effect on Gal4–Elk1 activity (Figure 5B). These results indicate that MAPK nuclear translocation is essential to maximally phosphorylate and activate one of its nuclear targets, Elk1. The inactive form of MKP-3 reduces the rate of S phase entry in response to mitogenic stimulation We then asked more generally whether the nuclear translo- cation of MAPK was necessary for the mitogenic response of the cells. To examine whether the prevention of MAPK nuclear translocation interferes with G /S phase progres- sion, we monitored DNA replication by bromodeoxyurid- ine (BrdU) incorporation. In contrast to cells expressing the PTP1C phosphatase, which apparently plays no role in S phase entry (Rivard et al., 1995), cells expressing WT MKP-3 were unable to enter S phase (Figure 6A and B). More importantly, cells expressing the inactive form of MKP-3 were also blocked in the G phase (Figure 6A and B). This suggested that the blockage of MAPK nuclear translocation prevented G /S phase progression. To exclude the possibility that MKP-3 could enter the nucleus transiently, as a shuttling molecule, we targeted MKP-3 C/S to the plasma membrane by generating a fusion protein with the sodium/proton exchanger (NHE– MKP-3 C/S). Ectopic expression of membrane-targeted MKP-3 C/S prevented MAPK nuclear translocation (data not shown) and also prevented DNA synthesis, whereas NHE alone did not (Figure 6A and B). Interestingly, similar results were obtained by expressing a version of NHE–MKP-3 in which we deleted the phosphatase cata- lytic domain, leaving intact the N-terminal MAPK-inter- Fig. 5. Inactive form of MKP-3 inhibits nuclear Elk1 activity. acting domain (Muda et al., 1998) (see Materials and (A) CCL39 cells were transiently transfected with the construct methods). This construct, when overexpressed, prevented encoding the Gal4–Elk1 fusion protein. The subcellular localization of this construct was assayed by immunofluorescence, using an anti-Elk1 MAPK nuclear translocation and G /S progression up to antibody. (B) CCL39 cells were transiently co-transfected with the 70% (data not shown). empty vector (pSG5), or vectors encoding MKP-3 (WT or the C/S As was the case for the luciferase activity (see Figure inactive mutant), Gal4–Elk1 fusion protein and the Gal4–luciferase 5B), the overexpression of p44MAPK reversed the reporter constructs. Vectors encoding HA-p44MAPK, HA-p38MAPK blockage of G /S phase progression due to the expression or HA-JNK were added at a 2:1 ratio compared with the MKP-3 1 vectors. Luciferase activity was assayed 2 days after transfection. The of MKP-3 inactive mutant but not that of the WT form values represent the mean  SEM of four independent experiments (Figure 6C). This reversion was specific to p44MAPK conducted in triplicate. and was not observed with either JNK or p38MAPK (Figure 6C). The inactive form of MKP-3 prevents nuclear Altogether, these experiments strongly suggest that Elk1-dependent transcription MAPK nuclear translocation is a crucial step in signal We asked whether the blockage of MAPK nuclear translo- transduction leading to gene expression and promoting cation affected the activation of MAPK-dependent nuclear cell cycle re-entry upon mitogenic stimulation. targets, such as transcription factors. To this end, we took advantage of the Gal4–Elk1 fusion protein which is Discussion localized predominantly in the nucleus (Figure 5A) and whose phosphorylation and subsequent activation by In this study, we have reported a means to specifically MAPK can be assayed using a Gal4–luciferase reporter prevent p42/p44MAPK nuclear translocation without gene. As expected, expression of MKP-3 WT which affecting its activation. Different methods can theoretically potently inactivates p42/p44MAPK (see Figure 1B) inhib- be used to achieve the blockage of MAPK nuclear translo- ited Gal4–Elk1 activation (Figure 5B). Interestingly, cation. The one we employed here was to create an expression of the inactive form of MKP-3 inhibited Gal4– artificial anchor for MAPK based on two criteria: a specific Elk1 activation by 60–70% (Figure 5B). The effect of interaction with MAPK and a cytoplasmic localization. MKP-3 C/S, but not that of MKP-3 WT, was reverted by Several proteins could possibly fulfil the criteria to create 669 A.Brunet et al. 670 Role of p42/p44MAPK nuclear translocation a cytoplasmic anchor for MAPK. For instance, the activator the endogenous form of MAPK (A.Brunet, unpublished of MAPK, MKK1, is a cytoplasmic protein that also binds observation). specifically to MAPK (Bardwell et al., 1996; Fukuda Therefore, preventing MAPK nuclear translocation by et al., 1997) and has therefore been proposed to play the anchoring it in the cytoplasm with an inactive phosphatase role of an MAPK anchor in vivo (Fukuda et al., 1997). represents an attractive method to assay the role of MAPK Moreover expression of MKK1 in Xenopus has been translocation. However, one potential drawback to this shown to impair MAPK nuclear translocation (Fukuda approach was that an efficient trapping of MAPK might et al., 1997). However, in our fibroblast CCL39 cell line, also prevent the accessibility to the upstream MKK1/2 or to the ability of MKK1 expression to prevent MAPK nuclear all the substrates. Our experiments, either using antibodies translocation was much weaker than that of inactive MKP- directed against the phosphorylated form of MAPK or 3. This can be explained by the fact that MKK1, when analysing the retardation in the electrophoretic mobility overexpressed, is less excluded from the nucleus than is of MAPK clearly showed that MAPK, in a complex with MKP-3, which is in agreement with the observation that MKP-3 C/S, was phosphorylated. This complexed MAPK MKK1 can enter the nucleus under certain circumstances was also active towards a substrate in vitro, as demon- (Jaaro et al., 1997). More importantly, MKK1 interacts strated with in vitro kinase assays (P.Lenormand, unpub- more weakly with MAPK than does MKP-3 (P.Lenormand, lished observation). As far as the accessibility to substrates unpublished observation). is concerned, we were able to show that when complexed Another method to prevent MAPK nuclear translocation to MKP-3, MAPK was still able to phosphorylate cyto- would be to affect its transport into the nucleus. The plasmic substrates in vivo. Therefore, the complex between mechanism by which MAPK is transported into the nucleus MKP-3 inactive mutant and MAPK appears to be tight is not yet known, but it has been hypothesized that enough to prevent MAPK nuclear translocation, but does transcription factors could co-transport with it, via a not affect MAPK general activation and substrate recogni- ‘piggy-back’ system. Recent experiments conducted with tion. The domains of MKP-3 involved in MAPK specificity Spc1, the p38MAPK homologue in fission yeast, show of recognition recently have been mapped in the N- that the deletion of the transcription factor Atf1, which is terminal region of the MKP-3 (Camps et al., 1998; Muda a major substrate of this kinase, prevented Spc1 relocaliz- et al., 1998). This domain alone, when targeted to the ation into the nucleus (Gaits et al., 1998). Previous work plasma membrane as a fusion with the NHE appears to from our laboratory has also supported the notion that be sufficient to prevent MAPK nuclear translocation. This nuclear proteins could play a role in MAPK nuclear result indicates that the cytoplasmic retention of MAPK translocation and retention (Lenormand et al., 1998). is mediated via the N-terminal MAPK-interacting site and However, interfering with transcription factors, which are is not as a result of ‘substrate trapping’ by the C/S mutant MAPK major targets, cannot be used to assay the role of of MKP-3. It would be interesting to determine the MAPK translocation in biological responses since these corresponding region of MAPK that interacts with MKP-3. responses may themselves require transcription. Since a mutant of MAPK resistant to MKP action previ- A third way to prevent MAPK nuclear translocation is ously had been mapped to the extreme C-terminal domain to act on MAPK itself. In a recent report, M.Cobb’s group of the MAPK (Bott et al., 1994; Camps et al., 1998), this showed that MAPK could form dimers when one of the region could be the one interacting with MKP-3. The fact monomers is phosphorylated and that this dimerization that the domains involved in the recognition of substrates could lead to MAPK nuclear translocation (Khokhlatchev were mapped in a more upstream domain of MAPK et al., 1998). Mutants of the C-terminal region of MAPK (Kallunki et al., 1994; Brunet and Pouysse´gur, 1996) which are unable to dimerize remain cytoplasmic even reinforces the observation that MAPK accessibility to upon growth factor stimulation (Khokhlatchev et al., substrate would not be impaired by its interaction with 1998). Since those mutants still retain their kinase activity, MKP-3. they could be used to assay the role of MAPK nuclear Using MKP-3 inactive mutant as a tool, we were able first translocation. Using a similar approach, we fused an NES to show that the blockage of MAPK nuclear translocation to the N- or C-terminal ends of p44MAPK in order to impaired MAPK-dependent activation of the transcription enforce its nuclear exit. However, these latter approaches factor Elk1. Although this result was expected, it had not rely both on the fact that MAPK dimer formation is yet been formally excluded that MAPK nuclear substrates efficient in vivo and that the introduction of a mutant form could have been phosphorylated in the cytoplasm, where of MAPK will also be able to prevent the endogenous they undergo obligatory transit during their biosynthesis. In form of MAPK from translocating into the nucleus, in a this alternative model, the relay between the cytoplasm and dominant-negative fashion. Preliminary results obtained the nucleus would have been established by transcription with the NES–MAPK indicate that these forms are not factors, as is the case for the JAK–STAT or the NF-κB able to function efficiently as dominant-negatives with pathways (Karin and Hunter, 1995). Since MAPK nuclear Fig. 6. Inactive MKP-3 mutant prevents S phase entry upon mitogenic stimulation. (A) CCL39 cells, either not transfected or transiently transfected with the expression vectors encoding PTP1C, MKP-3 (WT or C/S mutant), NHE or an NHE–MKP-3 C/S fusion construct were rendered quiescent for 24 h and stimulated for 24 h with 20% FCS, the BrdU being added for the last 4 h. Cells expressing the different constructs were revealed by immunofluorescence using polyclonal primary antibodies directed against each protein (left panel). Reinitiation of DNA synthesis was followed by BrdU incorporation, detected by an anti-BrdU monoclonal antibody (middle panel). The DNA was stained using DAPI (right panel). (B) Quanti- fication of the experiment presented in (A). Experiments were performed in duplicate, and 100 cells were counted for each condition. For PTP1C, MKP-3 WT and C/S, the values represent the mean and SEM of three independent experiments. (C) Expression vectors encoding HA-p44MAPK, HA-p38MAPK or HA-JNK were transfected in a 2:1 ratio compared with MKP-3. Experiments were conducted in the same way as in (A). Experiments were perfomed in duplicate and 100 cells were counted for each condition. 671 A.Brunet et al. rabbit antibodies were purchased from Sigma. Texas Red X-conjugated translocation is required for Elk1-dependent activation, our goat anti-mouse or anti-rabbit antibodies were from Molecular Probe. results suggest that the phosphorylation of Elk1 by MAPK The BrdU proliferation kit was purchased from Amersham. The MKK1 occurs in the nucleus. Moreover, the blockage of MAPK inhibitor PD 098059 was obtained from New England Biolabs. nuclear translocation inhibited the activity of the total c-fos Constructs promoter, confirming that MAPK entry into the nucleus The expression vectors encoding the Myc-tagged phosphatase Pyst1/MKP- is crucial for immediate early gene expression (A.Brunet, 3, either WT or an inactive mutant in which Cys293 of the catalytic site unpublished observation). Therefore, in the MAPK signal- has been replaced by a serine (C/S), were described previously (Groom ling pathway, the relay between the cytoplasm and the nuc- et al., 1996). The expression vectors encoding HA-tagged forms of leus appears to be constituted by MAPK itself. p44MAPK, p38MAPK (Brunet and Pouysse´gur, 1996), JNK1 (De´rijard et al., 1994) and p90RSK1 (Grove et al., 1993), the PTP1C phosphatase The approach of blocking the nuclear relocalization of (Rivard et al., 1995) and the human NHE1 (Sardet et al., 1989) have all MAPK could also be used to assay in which subcellular been described. The expression vectors encoding the Gal4–Elk1 fusion compartment different MAPK substrates are phosphoryl- protein and the Gal4–luciferase reporter gene have been described previ- ated. Another question that remains to be answered in terms ously (Janknecht et al., 1994) and were kindly provided by Dr K.Kelly. The vector encoding human Elk1 was kindly provided by Dr R.A.Hipskind. of compartmentalization is where the dephosphorylation The construct encoding a fusion protein between the NHE1 and the C- and consecutive inactivation of MAPK by the MKPs occurs terminal domain of Elk1 was generated by PCR, by inserting an ApaI in vivo. Observations made in Aplysia (Martin et al., 1997) restriction site at the level of amino acid 317 of Elk1 in-frame with the and in fibroblasts (P.Lenormand, in preparation) show that ApaI restriction site corresponding to amino acid 698 of NHE1. The after long-term stimulation, active MAPK is only found in construct encoding a fusion protein between NHE1 and MKP-3 C/S mutant was generated by PCR, by inserting an ApaI site at the level of the first the cytoplasm. Therefore, one important role for MAPK amino acid of MKP-3 C/S in-frame with the ApaI site corresponding to nuclear translocation could be to enter in contact with nuc- amino acid 698 of NHE1. The construct encoding a fusion protein between lear phosphatases (MKP-1 and -2 for instance) in order NHE1 and the N-terminal domain of MKP-3 was generated by PCR, using to shut down the signal. Endogenous MKP-3 could also the EcoRI site of MKP-3 and deleting the region encoding amino acids 230–359 of MKP-3, so that the last amino acid and the Myc epitope tag participate in signal extinction by dephosphorylating the were in-frame with the N-terminal domain of MKP-3. The constructs were cytoplasmic pool of MAPK. verified by sequencing. Finally, as far as biological responses are involved, our results show that MAPK nuclear translocation appears to Cell culture be required for progression into S phase in response to The Chinese hamster lung fibroblast cell line CCL39 (ATCC) or CCL39 expressing the chimeric Raf::ER chimeric construct (Lenormand et al., mitogenic stimulation. It would be interesting to test other 1996) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) biological responses that have been shown to be under the (Gibco-BRL) supplemented with 7.5% fetal calf serum (FCS; Biowhit- control of MAPK, such as differentiation of PC12 cells taker) and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) at (Traverse et al., 1992) or thymocytes (Alberola-Ila et al., 37°C in an atmosphere of 95% air, 5% CO . 1995; Crompton et al., 1996), survival of PC12 cells (Xia Immunofluorescence et al., 1995) or long-term potentiation in Aplysia (Martin CCL39 cells were plated onto glass coverslips at a density of 10 cells/ et al., 1997). Since most of them are dependent upon gene 35 mm dish. They were transfected by the calcium phosphate technique expression and protein synthesis, it seems likely that they with 7.5 μg of the different constructs. The day after transfection, cells will also be affected by MAPK retention in the cytoplasm. were rendered quiescent by incubation in serum-free medium for 16 h, and stimulated with various agonists. They were then fixed for 15 min at However, other MAPK-controlled responses, such as the –20°C with methanol/acetone (70:30 v/v) or methanol/formaldehyde (99:1 initiation of protein synthesis (Pause et al., 1994) or the v/v) or for 15 min in 10% formaldehyde at room temperature, followed by suppression of integrin activation (Hughes et al., 1997) may a methanol permeabilization for 10 min at –20°C for phospho-MAPK be independent of MAPK nuclear translocation. antibody. Coverslips were washed with phosphate-buffered saline (PBS) and the non-specific sites were blocked by incubation with PBS  3% In conclusion, our study provides a way to specifically bovine serum albumin (BSA). Coverslips were incubated with the first block MAPK nuclear translocation without affecting its antibody diluted in PBS/BSA (anti-HA, 1/500; anti-Myc, 1/100; anti- activation. In addition to demonstrating the importance of MAPK, 1/2000; anti-phosphoMAPK, 1/150; anti-p38MAPK, 1/300; anti- MAPK nuclear translocation for cellular processes such as JNK, 1/300; anti-Elk1, 1/500; anti-MKP-3, 1/100) for 1 h, then washed mitogen-induced gene expression and cell cycle progres- five times with PBS. Cells were incubated with the second antibody (goat anti-mouse FITC-conjugated, 1/100; goat anti-rabbit Texas Red- sion, this system should be useful for specifically blocking conjugated, 1/500) diluted in PBS/BSA for 1 h. After extensive washes in subsets of MAPK responses, an effect which is not possible PBS and in distilled water, coverslips were mounted in Mowiol (Calbi- when one interferes more generally with MAPK activation. ochem) and examined under epifluorescent illumination. BrdU incorporation CCL39 cells were transfected with the relevant constructs as described Materials and methods above. The day after transfection, cells were starved for 24 h and stimulated Material for 24 h with 20% FCS (Biowhittaker), with a pulse of BrdU (1/500) Monoclonal antibodies directed against the HA and Myc epitopes were for the last 4 h. Cells were fixed for 15 min at –20°C with methanol/ purchased respectively from Boehringer Mannheim and Santa Cruz. We formaldehyde (99:1 v/v) and incubated with primary antibody (anti-MKP- used commercially available polyclonal antibodies directed against 3, 1/100; anti-PTP1C; 1/250; anti-NHE, 1/500) prior to incubation with p42MAP kinase (Upstate Biotechnology), the dual-phosphorylated the secondary antibody (FITC anti-rabbit: 1/100) as described above. The p42MAPK (Promega), p38MAPK and JNK1/2 (Sigma), and p90RSK1 cells were then fixed in 3.7% formaldehyde for 10 min at room temperature, (Santa Cruz). Polyclonal antibodies directed against MKP-3/Pyst1 were rinsed in PBS and the chromatin was rendered accessible by a 10 min generated against the last 20 amino acids of the human isoform (Groom treatment with HCl (2 M). Cells were washed extensively with PBS and et al., 1996). The polyclonal antibodies against the PTP1C phosphatase blocked in PBS  10% FCS  0.1% Tween-20. The coverslips were were kindly provided by Dr E.R.Stanley (Rivard et al., 1995). The poly- incubated with the anti-BrdU monoclonal antibody (1/2) for 1 h, then clonal antibodies (RPc28) directed against the C-terminal region of the washed five times with PBS  0.1% Tween-20 (PBST). The secondary human NHE1 isoform were described previously (Sardet et al., 1990). The antibody (Texas Red anti-mouse, 1/500) was incubated for 45 min. DAPI purified CREB protein was kindly provided by Dr A.J.Shaywitz. (4,6 diamidine dihydrochloride; Boehringer Mannheim) was added for Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or anti- the last 15 min at a final concentration of 0.2 μg/ml. Cells were washed 672 Role of p42/p44MAPK nuclear translocation extensively in PBST then in distilled water and mounted in Mowiol (Cal- Alessi,D.R., Smythe,C. and Keyse,S.M. (1993) The human CL100 gene biochem). encodes a tyr/thr-protein phosphatase which potently and specifically inactivates MAP kinase and suppresses its activation by oncogenic ras Luciferase assays in Xenopus oocyte extracts. Oncogene, 8, 2015–2020. CCL39 cells were seeded in 24-well plates, at a density of 10 cells/well, Alessi,D.R., Cuenda,A., Cohen,P., Dudley,D.T. and Saltiel,A.R. (1995) and were co-transfected with 0.25 μg of the 5 Gal4–luciferase reporter PD 098059 is a specific inhibitor of the activation of mitogen-activated gene, 0.5 μg of the Gal4–Elk1 fusion construct, 0.5 μg of the the constructs protein kinase kinase in vitro and in vivo. J. Biol. Chem., 270, 27489– corresponding to MKP-3 and 1 μg of the constructs corresponding to the different MAPK family members. Two days after transfection, cells were Bardwell,L., Cook,J.G., Chang,E.C., Cairns,B.R. and Thorner,J. (1996) lysed in 100 μl of lysis buffer and the luciferase activity of one-tenth of Signaling in the yeast pheromone response pathway: specific and high- the samples was assayed according to the Promega protocol. affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7. Mol. Cell. Biol., Western blotting 16, 3637–3650. CCL39 cells expressing the Raf::ER chimera (Lenormand et al., 1996) Bott,C.M., Thorneycroft,S.G. and Marshall,C.J. (1994) The sevenmaker were seeded in 10 cm dishes at a density of 210 cells/dish. They were gain-of-function mutation in p42 MAP kinase leads to enhanced transfected by the SuperFect reagent (Qiagen) with 5 μg of the vector signalling and reduced sensitivity to dual specificity phosphatase. FEBS encoding the NHE–Elk1 fusion protein and 10 μg of the relevant constructs. Lett., 352, 201–205. The day after transfection, cells were submitted to an acid load selection Brondello,J.M., McKenzie,F.R., Sun,H., Tonks,N.K. and Pouysse´gur,J. (Pouysse´gur et al., 1984), in order to kill cells that did not express the (1995) Constitutive MAP kinase phosphatase (MKP-1) expression NHE–Elk1 protein. Resistant cells were rendered quiescent in serum-free blocks G specific gene transcription and S-phase entry in fibroblasts. medium for 5 h and stimulated with 30 nM estradiol for 30 min. Oncogene, 1085, 1895–1904. Membrane fractions were obtained by incubating the cells in a hypotonic Brondello,J.-M., Brunet,A., Pouysse´gur,J. and McKenzie,F.R. (1997) The buffer[10 mMTris–HCl pH8.0,1 mMEDTA,1 mMphenylmethylsulfonyl dual specificity mitogen-activated protein kinase phosphatase-1 and fluoride (PMSF), 1 mM phenanthroline, 1 mM iodoacetamide] for 10 min -2 are induced by the p42/p44MAPK cascade. J. Biol. Chem., 272, at 4°C. Cells were then scrapped and the membranes were pelleted by 1368–1376. centrifugation at 40 000 r.p.m. for 20 min at 4°C and directly resuspended Brunet,A. and Pouysse´gur,J. (1996) Identification of MAP kinase domains in 1 Laemmli sample buffer. Proteins were resolved on SDS–PAGE by redirecting stress signals into growth factor responses. Science, 272, (7.5%; 19:1 acrylamide/bisacrylamide) and transferred onto PVDF mem- 1652–1655. branes (Immobilon). The membranes were incubated with the anti-Elk1 Camps,M., Nichols,A., Gillieron,C., Antonsson,B., Muda,M., Chabert,C., antibody (1/1000) overnight at 4°C. Signal was detected using horseradish Boschert,U. and Arkinstall,S. (1998) Catalytic activation of the peroxidase (HRP)-conjugated anti-rabbit antibodies and the enhanced phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. chemiluminescencemethod. Cytoplasmicfractions wereresolvedon SDS– Science, 280, 1262–1265. PAGE (12.5%; 30:0.2 acrylamide/bisacrylamide) and immunoblotted with Cano,E. and Mahadevan,L.C. (1995) Parallel signal processing among the anti-p42MAPK antibody. mammalian MAPKs. Trends Biochem. Sci., 20, 117–122. Chen,R.H., Sarnecki,C. and Blenis,J. (1992) Nuclear localization and Kinase assay regulation of ERK- and RSK-encoded protein kinases. Mol. Cell. Biol., 293 cells were seeded in 35 mm dishes at a density of 750 000 cells per 12, 915–927. dish. They were transfected by the calcium phosphate technique with 5 μg Crompton,T., Gilmour,K.C. and Owen,M.J. (1996) The MAP kinase of the vector encoding HA-p90RSK1 and 1 μg of the vector encoding the pathway controls differentiation from double-negative to double- relevant constructs. positive thymocyte. Cell, 86, 243–251. At 24 h after transfection, cells were starved in serum-free medium for De´rijard,B., Hibi,M., Wu,I.-H., Barrett,T., Su,B., Deng,T., Karin,M. and 20 h, then stimulated with 20% FCS for 10 min. Cytoplasmic fractions Davis,R.J. (1994) JNK1: a protein kinase stimulated by UV light and were obtained by lysing the cells in a hypotonic buffer [5 mM Tris–HCl, Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell, pH 7.4, 1 mM MgCl 2 mM EGTA, 2 mM dithiothreitol (DTT), 5 mM 2, 76, 1025–1037. β-glycerophosphate, 1 mM PMSF, 1 mM aprotinin]. Extracts were Dounce Dudley,D.T., Pang,L., Decker,S.J., Bridges,A.J. and Saltiel,A.R. 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Journal

The EMBO Journal – Springer Journals

Published: Feb 1, 1999

Keywords: growth factors; MAP kinase; MAPK phosphatase; nuclear translocation; substrates

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