Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase, at Ser-446.

We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK >> p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK.

Mitogen-activated protein kinases (MAPKs) 1 are critical components of signal transduction pathways and mediate the cellular response to numerous extracellular stimuli, ranging from growth factors to environmental stress (1)(2)(3)(4). Five MAPK pathways have been identified in eukaryotic cells, of which three are relatively well characterized: the extracellular signal-regulated kinase (ERK) pathway, the c-Jun amino-terminal kinase (JNK) pathway, and the p38 pathway. The activation of MAPK is strictly regulated by two upstream kinases, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK). MAP-KKKs, such as Raf, MEKK, MLK, and ASK1, phosphorylate and activate MAPKK. MAPKK is a dual-specificity protein kinase that phosphorylates tyrosine and threonine residues in a TXY motif of MAPK, leading to the full activation of MAPK (5). Once activated, MAPK can translocate from the cytoplasm to the nucleus, leading to phosphorylation of several transcription factors and altered regulation of gene expression (6,7). The physiological functions of various MAPK subfamilies have been studied extensively in many systems. In general, activation of the ERK cascade leads to cell proliferation, differentiation, and enhanced cell survival after cellular stress, although in certain situations, ERK activation is required for the execution of apoptosis (8,9). On the other hand, activation of the JNK and p38 cascades are usually associated with enhanced apoptosis and production of inflammatory cytokines (3, 10 -12), although there are notable exceptions in which JNK/p38 is necessary for cell proliferation and differentiation (13,14). Because MAP kinase pathways play important roles in regulating many critical cellular processes, the precise regulation of these signaling proteins is crucial for maintenance of cellular homeostasis.
MAPK pathways are regulated at multiple levels to ensure the specificity, timing, and strength of their action (6). One critical aspect of this regulation is reversible phosphorylation of MAPKs. Negative regulation of MAPKs is achieved by dephosphorylation of the TXY motif by phosphatases (5,15). As an in vivo candidate for negative regulator, the MAPK phosphatases (MKPs), a family of dual-specificity protein phosphatases, has emerged (16,17). In mammals, 10 MKPs have been reported, and they are precisely regulated in their substrate specificity to avoid inappropriate inactivation of MAPKs (16,17). MKPs are primarily composed of two domains, a rhodanese-like domain and a dual-specificity phosphatase catalytic domain (18). Recently we and other groups identified MKP-7, which possesses a long COOH-terminal stretch (CTS) containing both a nuclear export signal (NES) and a nuclear localization signal, in addition to the rhodanese-like domain and the dual-specificity phosphatase catalytic domain (19,20).
MAPKs interact with MAPKKs, MAPK substrates, and phosphatases, and the specificity of each interaction likely regulates the enzymatic efficiency and specificity of the MAPK pathways. Recently two regions, named the CD and ED sites, have been identified on the surface of MAPKs and shown to act as docking sites for complex formation (21,22). Both sites contain negatively charged clusters of residues. How MAPK interacting proteins bind MAPK through the CD or ED sites is not yet clear. Recently, the presence of common MAPK docking domains were proposed in MAPKKs (21,23,24), MKPs (25)(26)(27)(28)(29), and MAPK substrates such as transcription factors (30 -38) and downstream kinases (22,39,40). The MAPK-docking domain (D-domain) is composed of three submotifs: a cluster of basic residues, an LXL element, and a cluster of hydrophobic residues.
In a previous study (19), we identified MKP-7 as a JNKspecific phosphatase (JNK Ͼ Ͼ p38 Ͼ ERK) with the characteristics of a nucleocytoplasmic shuttle protein. Furthermore, we demonstrated that MKP-7 bound ERK2 and p38␣ as well as JNK1 despite its high specificity toward JNK1 as a substrate. This observation suggested that binding is necessary but not sufficient for determination of substrate specificity, and that there is an unidentified function associated with interaction between MKP-7 and ERK2. Here, we show that activation of ERK induces an upward mobility shift of MKP-7 on SDS-PAGE gels, resulting from phosphorylation. We also analyze the function of the CTS in interacting with MAPKs and inactivating them, and the function of the two putative D-domains, designated D-domain I and D-domain II. We found that the CTS is a critical determinant of substrate specificity and a target of ERK, which was suggested to phosphorylate MKP-7 on Ser-446. Finally we present evidence that D-domain II is important for its catalytic activity as a MAPK phosphatase.
Detection of Expressed Proteins-Methods for preparation of cell lysates and for immunoprecipitation were as described previously (19). Each sample was separated by a SDS-PAGE on 7.5 or 10% gels and transferred to a nitrocellulose membrane. The expression level of HAtagged MAPKs and FLAG-tagged MKP-7 wild type or mutant proteins was monitored by an anti-HA (12CA5) monoclonal antibody (Roche Diagnostics) and an anti-FLAG M2 monoclonal antibody (Sigma), respectively. The activation of ERK2, JNK1, and p38␣ was monitored by an anti-phospho-ERK antibody (New England Biolabs), an anti-ACTIVE JNK antibody (Promega), and an anti-phospho p38 antibody (New England Biolabs), respectively. Signals were detected by enhanced chemiluminescence using the ECL reagent (Amersham Biosciences).
Alkaline Phosphatase Treatment-Immunoprecipitates of FLAG-MKP-7WT or FLAG-MKP-7CS by an anti-FLAG M2 monoclonal antibody were suspended in phosphate-buffered saline and divided into five aliquots, one of which received 1ϫ SDS sample buffer. Two aliquots were washed once with calf intestine alkaline phosphatase (CIAP) reaction buffer (TaKaRa), and the resulting precipitates were resuspended in CIAP buffer with or without 20 units of CIAP (TaKaRa). The remaining 2 aliquots were similarly washed with bacterial alkaline phosphatase (BAP) reaction buffer (TaKaRa), and the precipitates were resuspended in BAP buffer with or without 20 units of BAP (TaKaRa). The dephosphorylation reaction was performed at 37°C for 30 min and terminated by addition of 5ϫ SDS sample buffer and boiling for 3 min.

RESULTS
Detection of Modified Forms of MKP-7-We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK1 Ͼ Ͼ p38␣ Ͼ ERK2 (19). However, MKP-7 can bind ERK2 as well as JNK1 and p38␣. To determine the physiological consequence of MKP-7 and ERK interaction, we co-expressed FLAG-MKP-7 with HA-ERK2 in COS-7 cells (Fig. 1). We found that the band representing FLAG-MKP-7WT (wild type) was shifted upward dependent on PMA stimulation when HA-ERK2 was co-expressed. To deter- suggesting that activation of HA-ERK2 following PMA treatment is associated with modification of FLAG-MKP-7, and that MKP-7 enzymatic activity antagonizes this effect. When HA-JNK1 was co-expressed, modification of FLAG-MKP-7WT following sorbitol stimulation was not observed (Fig. 1b, lane 6). However, a slight band shift of the CS mutant protein was observed with activation of JNK upon sorbitol stimulation (Fig.  1b, lanes 7 and 8). Co-expression of HA-p38␣ did not induce modification of either the wild type protein or the CS mutant of FLAG-MKP-7. These results indicate that modification of FLAG-MKP-7 is correlated with the activation of HA-ERK2, and that the catalytic activity of FLAG-MKP-7 antagonizes such modifying activity.
Modification of MKP-7 Is Regulated by Activated HA-ERK Activity-To clarify whether MKP-7 modification is because of activated ERK, we observed the effect of U0126, a MEK inhibitor, on the upward mobility shift of MKP-7. The phosphorylation level of HA-ERK2 was increased by PMA and was decreased with U0126 treatment in COS-7 cells expressing HA-ERK2 and FLAG-MKP-7WT or HA-ERK2 alone (Fig. 2). Under these conditions, modification of MKP-7 was diminished by U0126 treatment (Fig. 2 lanes 6 -10). When FLAG-MKP-7WT was expressed alone in COS-7 cells, PMA did not induce the upward mobility shift at all (Fig. 2, lanes 1-5). These results demonstrate that the observed modification of FLAG-MKP-7WT was induced by co-expression of activated HA-ERK2.
FLAG-MKP-7 Protein Is Phosphorylated following PMA Stimulation-Slower migrating forms of FLAG-MKP-7WT could result from post-translational modification such as phosphorylation. Therefore, we determined whether the shifted band was susceptible to phosphatase treatment. FLAG-MKP-7WT or CS was co-expressed with HA-ERK2 in COS-7 cells and immunoprecipitated with anti-FLAG antibody. The immunocomplex was then incubated with CIAP or BAP (Fig. 3). The shifted band collapsed into a slightly smaller band following CIAP or BAP treatment, but was not affected by treatment with the corresponding buffer ( Fig. 3A, lanes 10 -14). The shifted band of FLAG-MKP-7CS also collapsed by CIAP and BAP treatment (Fig. 3B, lanes 10 -14). Such an effect by alkaline phosphatase was blocked by a phosphatase inhibitor, sodium pyrophosphate (data not shown). These results indicate that the shifted band of FLAG-MKP-7WT and FLAG-MKP-7CS corresponds to phosphorylated forms of the protein.
The COOH-terminal Region of MKP-7 Is Phosphorylated by ERK-To identify phophorylation site(s) of MKP-7, we used several FLAG-MKP-7 deletion mutants such as FLAG-MKP-7-delC2, -delC3, and -delC4 shown in Fig. 6A. Expression vectors encoding the mutants were co-transfected with HA-ERK2 into COS-7 cells, and the cells were stimulated with PMA. As shown in Fig. 4A, modification of FLAG-MKP-7WT, -delC3, and -delC4 was observed, but the delC2 protein was not modified, strongly suggesting that phosphorylation site(s) are present in residues 371-568. To determine which amino acid is phosphorylated, the same blot used in Fig. 4 was incubated with antiphosphotyrosine or anti-phosphothreonine antibodies. No signals were detected at the position corresponding to the shifted band (data not shown), suggesting that a serine residue(s) in residues 371-568 is potentially phosphorylated upon HA-ERK2 activation.
Serine 446 Is the Phosphorylation Site Dependent on ERK Activation-We searched for an S-P motif, a putative ERK phosphorylation site (41,42), in residues 371-568 of human MKP-7, and, as shown in Fig. 4B, found four SP motifs con- served in human and mouse MKP-7. To identify the phosphorylation site(s) in vivo, we generated a series of SA mutants, each with a single Ser to Ala substitution, namely, S374A, S446A, S459A, and S501A. FLAG-MKP-7WT or each SA mutant was co-expressed with HA-ERK2 in COS-7 cells. As shown in Fig. 4C, the bands representing FLAG-MKP-7 S374A, S459A, and S501A were shifted upward following HA-ERK2 activation to a level similar to the wild type protein; however, the band representing FLAG-MKP-7 S446A was not shifted under conditions in which HA-ERK2 was strongly activated (Fig. 4C, lane 8). These results clearly show that Ser-446, which is located in PEST sequences, is the site of phosphorylation in vivo upon ERK activation, and that this phosphorylation represents the modification observed in MKP-7.
Putative MAPK-binding Sites in MKP-7-Because Ser-446 is a putative ERK phosphorylation site and phosphorylation was inhibited by U0126, it is likely that ERK2 recognizes MKP-7 as a substrate. To determine how MKP-7 recognizes JNK as a substrate and how MKP-7 is recognized by ERK as a substrate, we analyzed the region necessary for binding to MAPKs in MKP-7. To search for the MAPK-docking domain (D-domain) in MKP-7, we aligned the primary sequence of MKP-7 with the D-domain of MAPKKs (21,23,24) or transcription factors, which are targets of MAPKs, such as c-Jun (31,32,38), Elk-1 (34,35,43), SAP-1 (33,36), and MEF2A/2C (30,33,37) (Fig.  5A). We found two D-domains in MKP-7 at residues 52-69 in the rhodanese-like domain and in residues 161-180 in the DSP catalytic domain, and designated them D-domains I and II, respectively. The D-domain I exhibits conserved basic and hydrophobic motifs, whereas D-domain II has conserved LXL and hydrophobic motifs. Both D-domains are highly conserved among MKPs (Fig. 5B). To determine the potential function of these domains in interaction with the three MAPKs, we introduced several mutations into these domains (Fig. 6A).
Role of D-domains in Interaction with JNK and p38 -It has been proposed that the basic cluster in several transcription  factors, such as MEF-2A/2C, SAP-1, and Elk-1, which also occurs in D-domain I, is important for binding to a MAPK (30,(33)(34)(35)(36)(37). Because it was reported that basic residues, Arg or Lys, to Ala substitutions at the basic cluster in MEF-2A/2C, SAP-1, and Elk-1 disrupts binding to MAPKs. Therefore, we introduced mutations in Arg-56 and Arg-57 of MKP-7 as shown in Fig. 6A and examined whether the D-domain I is required for interaction with MAPKs. The interactions of various MKP-7 mutants with three MAPKs were analyzed by immunoprecipitation. In COS-7 cells, FLAG-MKP-7R2A was expressed at levels comparable with FLAG-MKP-7WT (Fig. 6B, lanes 5 and  6). Although amounts of co-immunoprecipitated HA-ERK2 and HA-JNK1 with FLAG-MKP-7R2A were comparable with those seen with FLAG-MKP-7WT, HA-p38␣ was not co-precipitated with FLAG-MKP-7R2A, suggesting that D-domain I in MKP-7 is essential for interaction with p38␣ but not with ERK2 or JNK1.
It has been also proposed that the LXL element in several transcription factors such as c-Jun, SAP-1, and Elk-1 (Fig. 5A), which also occurs in D-domain II, is important for binding to MAPK (33,34,43). Because it was reported that Leu to Ala substitutions at the LXL element of the MAPK-docking domain in c-Jun, SAP-1, and Elk-1 disrupts binding to MAPKs, we introduced mutations in Leu-166 and Leu-168 as shown in Fig.  6A and analyzed interaction with three MAPKs in COS-7 cells (Fig. 6B, lanes 7 and 8). The amount of HA-ERK2 co-immunoprecipitated with FLAG-MKP-7L2A was comparable with that of FLAG-MKP-7WT. However, the amount of co-immunoprecipitated HA-JNK1 and HA-p38␣ with FLAG-MKP-7L2A was reduced compared with that seen with FLAG-MKP-7WT. Such interaction with JNK and p38 was also obvious using COOHterminal deletion mutants as shown in Fig. 6D (see b, lanes 7  and 8, and c, lanes 7 and 8). These results suggest that Ddomain II in MKP-7 is important for interaction with JNK1 and p38␣. It is noteworthy that HA-ERK2 interacted with 3 mutant proteins (the R2A, L2A, and RL4A) as did the WT protein, suggesting that neither D-domain I nor D-domain II in MKP-7 is required for ERK binding.
The Role of the Long COOH-terminal Stretch for Its Interaction with ERK-As MKP-7 possesses a unique CTS in addition to the rhodanese-like domain and DSP-catalytic domain (Fig.  6A), it was possible that the CTS interacts with the three MAPKs, particularly with ERK. We therefore analyzed whether the CTS is important in MAPK binding (Fig. 6C). When FLAG-MKP7-delC2, which is deleted in the CTS from residues 371 to 665, termed CTS-C2, was co-expressed with HA-MAPKs in COS-7 cells, similar levels of HA-ERK2, HA-JNK1, and HA-p38␣ were precipitated with FLAG-MKP-7-delC2 (Fig. 6C).
To test whether the CTS-C2 cooperates with either D-domain I or D-domain II in interaction with MAPKs, we introduced R2A or L2A mutations into the delC2 mutant protein. FLAG-MKP-7-delC2, -delC2/R2A, -delC2/L2A, or -delC2/R2E (opposite charge substitution at Arg-56 and Arg-57) was coexpressed with HA-MAPKs (Fig. 6D) in COS-7 cells. FLAG-MKP-7-delC2/R2A and -del2/R2E mutant proteins lost the interaction with HA-ERK2 (Fig. 6D, a, lanes 5 and 6 and 9 and   10), suggesting that D-domain I together with the CTS-C2 is essential for ERK binding. Compared with FLAG-MKP-7 L2A, interaction of FLAG-MKP-7-delC2/L2A with HA-JNK1 was more impaired (Fig. 6D, b, lanes 7 and 8), suggesting that D-domain II together with the CTS-C2 is important for the interaction with JNK1. By contrast, for complex formation with p38␣ the CTS-C2 is dispensable (compare Fig. 6B, panel c, with  D, panel c). The results shown in Fig. 6, B-D Importance of D-domain II and the CTS in Recognition of MAPKs as Substrates-We then determined which domains were required for inhibition of MAPK activation (Fig. 7). As we have reported, the inhibitory activity of FLAG-MKP-7 on HA-JNK1, HA-p38␣, and HA-ERK2 activation is strong, weak, and very weak, respectively (Fig. 7A, b, lane 6). FLAG-MKP-7R2A showed similar inhibitory activity as the wild type protein, but the L2A and RL4A mutants did not inhibit JNK activation, suggesting that D-domain II rather than D-domain I is important to inhibit JNK activation (Fig. 7A, b, lanes 10 and 12).
It was noteworthy that deletion of the CTS-C2 allowed inhibitory activity of FLAG-MKP-7 on HA-ERK2 to become apparent (Fig. 7B, a, lane 4). Likewise, deletion of the CTS-C2 significantly increased MKP-7 inhibition of HA-p38␣ (Fig. 7B,  c, lane 4). These observations suggest that the CTS-C2 of MKP-7 interferes with recognition of HA-ERK2 and especially HA-p38␣ as substrates. Then involvement of D-domain I and D-domain II in inhibition of MAPKs was analyzed using delC2 mutants (Fig. 7B). The D-domain II was confirmed to be an important domain for inhibition of JNK activation (Fig. 7B, b,  lane 8). L2A mutation impaired more than R2A or R2E mutation on the delC2 mutation on the activity of the delC2 mutant on HA-p38␣ activation (compare Fig. 7B, c, lane 8 with lanes 6  and 10), suggesting that D-domain II plays a more important role in inhibiting p38 activation than D-domain I. Furthermore, L2A mutation in the delC2 mutant impaired ERK inactivation activity without affecting interaction with ERK. These results strongly suggested that D-domain II plays an important role in recognition of MAPKs as substrates.
Association with the CTS Is Required for Phosphorylation of MKP-7 by ERK-We then analyzed the sequence required for phosphorylation of MKP-7 by ERK. Phosphorylation was not blocked by mutations in either D-domains I or II, or in both (Fig. 7A, a). These observations suggested that an association of ERK with the CTS, neither with D-domain I nor II, is essential for phosphorylation.
Interestingly, like the CS (phosphatase dead) mutant shown in Fig. 1b, the L2A and RL4A mutants, both of which lost inhibitory activity toward JNK activation, were modified following activation of HA-JNK1 (Fig. 7A, b, lanes 10 and 12). Because modification of the CS, L2A, and RL4A mutants was not inhibited by U0126 (data not shown), it is possible that JNK can phosphorylate the CTS only when the MKP-7 activity is impaired. These data suggested that HA-ERK2 but not HA- Effects of Several Stimuli on MKP-7 Phosphorylation-We asked if phosphorylation of FLAG-MKP-7 is induced by a variety of stimuli, including FBS, EGF, sorbitol, NaCl, H 2 O 2 , UV-C, and ionomycin. As shown in Fig. 8, EGF, PMA, UV-C, and ionomycin induced significant activation of HA-ERK2, resulting in the modification of FLAG-MKP-7WT (Fig. 8,  lanes 3, 4, 7, and 8). Stimulation by FBS and H 2 O 2 induced a slight activation of HA-ERK2 with a slightly shifted band of FLAG-MKP-7 (Fig. 8, lanes 2 and 7). Stimulation by sorbitol and NaCl induced a slight activation of HA-ERK2, but a shifted band was not detected (Fig. 8, lanes 5 and 6). The upward mobility shifts observed here were diminished by U0126 treatment (data not shown). These data indicate that external stimuli activate HA-ERK2, which induced modification of FLAG-MKP-7, likely the result of phosphorylation, and such phosphorylation levels are positively correlated with the extent of HA-ERK2 activation. DISCUSSION In the present study, we found that upon PMA stimulation activated HA-ERK2 induced an upward mobility shift of FLAG-MKP-7 on SDS-PAGE. The results that the upward mobility shift was reduced by alkaline phosphatase treatment indicated that modification was because of phosphorylation. Using U0126 and several mutant forms of MKP-7, we identified Ser-446 as the site phosphorylated in the presence of activated ERK (Fig. 9A). We analyzed the MKP-7 sequences required for interaction with MAPKs and inhibition of MAPK activation and made several observations (Fig. 9, B and C We have demonstrated the molecular basis of MKP-7 as the JNK-specific phosphatase. Importantly, FLAG-MKP-7-delC2 strongly inhibited p38␣ relative to JNK1, suggesting that the CTS from residues 371 to 665 determines JNK1 preference of MKP-7 by masking its activity on p38␣. Further analysis of the three-dimensional structure of the CTS will be required to determine the structural basis for this selectivity. It is important to note that another JNK phosphatase, hVH-5 (44), also has a long COOH-terminal sequence that is 30% homologous with the MKP-7 CTS. It is possible that the JNK preference of hVH-5 is also determined by the COOH-terminal sequences.
We found that the interaction of MKP-7 through D-domain II is indispensable in inhibiting JNK1 activation, and the Ddomain II is more important than D-domain I in inhibiting p38␣ activation (Fig. 9C). Although inhibition activity of MKP-7 on ERK2 activation was small, D-domain II as well as the D-domain I were important. Recently we and others demonstrated that VHR, a small molecular weight dual specificity protein phosphatase, binds ERK and inhibits activation of ERK, JNK, and p38, although it lacks the rhodanese-like domain (45)(46)(47)(48). As shown in Fig. 5B, VHR exhibits a single conserved D-domain corresponding to the position of domain II of MKPs. It is likely that domain II of MKPs is essential to inhibit MAPK activation. Recently another small molecular weight dual specificity protein phosphatase, LMW-DSP2, was identified and shown to contain a single D-domain homologous to domain II. Interestingly it was shown to inactivate p38 and JNK but not ERK (49). Taken together it is likely that substrate specificity may be embedded in D-domain II.
Phosphorylation of FLAG-MKP-7 in HA-ERK2 overexpressing cells was also induced by stimulation with several agents including FBS, EGF, H 2 O 2 , UV-C, and ionomycin, but not by osmotic stresses such as 0.4 M sorbitol and 0.5 M NaCl treatments (Fig. 8). The level of the phosphorylation of FLAG-MKP-7 was positively correlated to HA-ERK2 activation irrespective of the stimulus, supporting our conclusion that activated ERK is directly involved in the phosphorylation of MKP-7. However, it should be noted that 0.4 M sorbitol, which induces activation of HA-ERK2 comparable with 0.1 mM H 2 O 2 , did not induce MKP-7 phosphorylation. This finding could be because of dephosphorylation of phospho-Ser-446 by a Ser/Thr protein phosphatase that has been reported to be activated by osmotic stress (50).
Co-expressed HA-JNK1 did not induce modification of FLAG-MKP-7WT. However, it is noteworthy that FLAG-MKP-7CS, a phosphatase dead mutant, was modified in HA-JNK1 overexpressing cells (Fig. 1b). FLAG-MKP-7L2A, which lost inhibition activity toward HA-JNK1 activation because of a mutation at D-domain II, was also modified when co-expressed HA-JNK1 was activated (Fig. 7A, b). As it was demonstrated that JNK interacts with the CTS of MKP-7, these data suggest that when MKP-7 is impaired in its ability to inhibit JNK activation, activated JNK can phosphorylate Ser-446 (Fig. 9A). It is of interest to determine whether JNK phosphorylates MKP-7 in vivo.
So far some MKPs have been reported to be phosphorylated. MKP-1 is phosphorylated by ERK1/2 in vivo as well as in vitro protecting from protein degradation (51). hVH-5 is phosphorylated in response to PMA treatment, but the physiological consequences of such modifications are not known (52). Xenopus CL100 (XCL100), a homologue of human MKP-1, is phosphorylated by ERK in a cell cycle-dependent manner. In the case of XCL100, serine residue(s) were phosphorylated during the G 2 phase, and serine and threonine residues were phosphorylated during M-phase (53). Although the biological consequence of MKP-7 phosphorylation is currently unknown, it is important that Ser-446 in the CTS is a phosphorylation site. As we have previously demonstrated, the CTS is required for nucleocytoplasmic shuttling (19). Data presented here also shows that the CTS determines substrate specificity. Analyses of how phosphorylation affects substrate specificity and subcellular localization of MKP-7 are in progress. In addition, it is important to examine whether endogenous MKP-7 is phosphorylated as well. To address this question, we are preparing an antibody against phospho-Ser-446 of MKP-7. The present study suggests that MKP-7 could link ERK activity to stress kinases activation. Recently, it was reported that stress stimuli lead to an inhibition of ERK1/2 via p38 (54). Cross-talk between ERK and stress kinases such as JNK and p38 is an issue to be clarified.
Because MKPs are potential negative regulators of the MAPK cascade, they could play a role in carcinogenesis by regulating cell proliferation and apoptosis. We previously reported that overexpression of MKP-2 mRNA is tumor-specific in hepatocarcinogenesis (55). MKP-7 is thought to be a candidate tumor suppressor, because human MKP-7 maps to ch12p12, an area prone to deletions in several hematological malignancies of both myeloid and lymphoid organs and in solid tumors (19,56). It has been reported that JNK is constitutively activated in several tumor cell lines and that the transforming action of some oncogenes is JNK dependent. Expression of MKP-7 in tumors where MKP-7 could be tumor suppressive and may represent a potential therapeutic approach to some malignancies.