The JNK-interacting Protein-1 Scaffold Protein Targets MAPK Phosphatase-7 to Dephosphorylate JNK*

Emma A. WilloughbyDagger §, Gordon R. PerkinsDagger §, Mary K. CollinsDagger , and Alan J. Whitmarsh||**

From the Dagger  Department of Immunology and Molecular Pathology, University College London and Royal Free Medical School, Windeyer Institute, London W1T 4JF, United Kingdom and the  School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom

Received for publication, July 22, 2002, and in revised form, January 9, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) are activated by pleiotropic signals including environmental stresses, growth factors, and hormones. A subset of JNK can bind to distinct scaffold proteins that also bind upstream kinases of the JNK pathway, allowing sequential kinase activation within a signaling module. The JNK-interacting protein-1 (JIP-1) scaffold protein specifically binds JNK, MAP kinase kinase 7, and members of the MLK family and is essential for stress-mediated JNK activation in neurones. Here we report that JIP-1 also binds the dual-specificity phosphatases MKP7 and M3/6 via a region independent of its JNK binding domain. The C-terminal region of MKP7, homologous to that of M3/6 but not other DSPs, is required for interaction with JIP-1. When MKP7 is bound to JIP-1 it reduces JNK activation leading to reduced phosphorylation of the JNK target c-Jun. These results indicate that the JIP-1 scaffold protein modulates JNK signaling via association with both protein kinases and protein phosphatases that target JNK.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The JNK1 group of MAPKs are important for many cellular responses including apoptosis, growth, differentiation, embryonic development, and the immune response (1). In particular, JNK mediates the cellular response to stress signals (1). Similar to other MAP kinases, JNK is activated by phosphorylation by a signaling module that consists of a MAP kinase kinase (MKK) and a MAP kinase kinase kinase (MKKK) (1). Components of JNK-signaling modules include the MKKs MKK4 and MKK7, together with many types of MKKKs including MEKKs, MLKs, and apoptosis signal-regulating kinase 1 (1). Once activated, JNK phosphorylates and regulates the activity of a number of transcription factors including the activator-protein 1 family members c-Jun and activating transcription factor 2 (1).

MAPK activity is also regulated by protein phosphatases (2). In unstimulated cells JNK phosphatase activity is required to counteract the basal level of MKK4 and MKK7 activity and suppress JNK activation, whereas following cell stimulation JNK phosphatases can down-regulate JNK activity to basal levels (3-5). Inhibitor studies suggest that tyrosine, serine/threonine, and dual-specificity phosphatases contribute to JNK phosphatase activity (4). A large family of dual-specificity phosphatases (DSPs) has been identified that appear highly specific for MAPKs (2, 6). Over-expression studies have suggested that different dual-specificity phosphatases display different activities toward the MAPKs JNK, p38, and ERK. These experiments show that M3/6 (7), MKP5 (8, 9), and MKP7 (10-12) have higher activity against JNK than p38 or ERK, whereas MKP3 (13), MKP4 (14), and PAC1 (15) appear more specific for ERK. MKP1 appears to have equal activity against JNK, p38, and ERK (16).

Scaffold proteins bind components of MAPK-signaling modules and regulate their activity and intracellular location (17, 18). JNK signaling is controlled by scaffold proteins including both the JIP family and beta -arrestin-2 (19-26). JIP-1 and JIP-2 share extensive sequence homology and are mainly expressed in neuronal tissues, testis, and beta -cells; however, lower levels are present in many cell types (19, 20, 22). They interact with JNK, MKK7, and MLKs and can enhance signaling by this MAPK module (21, 22). JIP-2 has also recently been reported to interact with isoforms of p38 MAPK (27, 28). The third member of the family, JIP-3, displays no significant sequence homology with JIP-1 or JIP-2, but it also binds to multiple components of JNK-signaling modules (23, 24). In addition to the MLK-MKK7-JNK-signaling module, a number of other proteins have been demonstrated to interact with JIP-1, including hematopoietic progenitor kinase 1 (21), RhoGEF190 (29), beta -amyloid precursor protein (30), kinesin light chain (26), and apolipoprotein E receptor-2 (31). The physiological functions of these interactions with JIP-1 remain to be elucidated. Genetic studies indicate a role for JIP-1 in neuronal apoptosis. The targeted deletion of the Jip1 gene in mice results in reduced apoptosis of hippocampal neurones in response to stress, which coincides with reduced JNK activity and reduced phosphorylation of the JNK target c-Jun (32). It has also been reported that JIP-1 may suppress JNK activation in some cell types (33, 34) or play a role in vesicle trafficking (26). In this study we sought to determine whether, in addition to recruiting protein kinase components of JNK-signaling modules, JIP scaffold proteins could recruit DSPs to regulate JNK signaling.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Plasmids-- MKP2, MKP4, MKP7, and PAC1 were amplified by PCR using Turbo PFU (Stratagene) from expressed sequence tags (Integrated Molecular Analysis of Genomes and their Expression (IMAGE) Consortium identification numbers: 3605895, 3501447, 4400399, and 4297852) and then subcloned into the vectors pcDNA4HIS (Invitrogen) and fused to an N-terminal Xpress/T7 epitope tag or into pcDNA3.1 lacking an epitope tag (Invitrogen). MKP7 deletion constructs were subcloned into pcDNA4HIS fused to an N-terminal Xpress/T7 epitope tag. pMT M3/6 was a gift from Professor Alan Ashworth (Institute of Cancer Research, London, UK). HA-JNK3 was a gift from Dr. Julian Downward (Cancer Research, London, UK), and was subcloned into pcDNA3.1. Mutation of Cys-244 in MKP7 to Ser was performed using the oligonucleotide primer 5'-GATGTGTTCTAGTGCACAGTTTAGCCGGGATCTCC', together with a complementary oligonucleotide and the QuikChangeTM kit from Stratagene. Plasmids expressing T7-JIP-1 (21), T7-JIP-2 (22), T7-JIP-3 (23), GST-JIP-1, and JIP-1 deletions (21), HA-JNK2 (22), and HA-MLK3 (21) have been described previously. The construct expressing HA-c-Jun was provided by M. Karin (University of California, San Diego).

Production of Anti-MKP7 Polyclonal Antiserum-- Residues 345-665 of MKP7 were expressed from the vector pET28 in the bacterial strain BL21 (DE3). Cell extracts were prepared in 50 mM Tris, pH 8.8, 300 mM NaCl, and 0.5% IGEPAL CA-630, and centrifuged at 14,000 × g for 10 min. The supernatant was discarded and the pellet resuspended in 10 mM Tris, pH 8.0, 8 M urea, and 100 mM NaH2PO4. The MKP7 fragment was bound to Ni-agarose (Quiagen) then eluted in 10 mM Tris, pH 4.5, 6 M urea, and 100 mM NaH2PO4. The final pH was adjusted to pH 7.0, and a rat was immunized at three weekly intervals. The first immunization of 50 µg of MKP7 and Complete Freund's Adjuvant was via the Peyers Patches after surgical exposure. The second immunization was also with 50 µg of MKP7 into the Peyers Patches but Incomplete Freund's Adjuvant was used. The third immunization was done intraperitoneally with 80 µg of MKP7 mixed with Incomplete Freund's Adjuvant. The final immunization was again via the Peyers Patches with 50 µg of MKP7 and Incomplete Freund's Adjuvant. 3 days later the rat serum was collected and used unpurified at a dilution of 1/2000 for immunoblotting or 1/500 for immunoprecipitation.

Cell Culture-- 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. ND7 and N1E-115 cells were maintained in DMEM supplemented with 10% fetal bovine serum. COS-7 cells were maintained in DMEM supplemented with 5% fetal bovine serum. COS-7 and 293T cells were transfected using LipofectAMINE (Invitrogen) according to the manufacturer's instructions.

Pull Downs, Immunoprecipitations, and Immunoblots-- 293T cells were washed once with ice-cold phosphate-buffered saline then lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% IGEPAL CA-630, 5 mM EDTA, and a protease inhibitor mixture (Roche Molecular Biochemicals). After incubation on ice for 10 min, extracts were centrifuged at 14000 × g for 10 min. Supernatants were recovered and combined with an appropriate volume of gel loading buffer (final concentration 50 mM Tris, pH 6.8, 2% SDS, 100 mM dithiothreitol, 4% glycerol) or subject to pull down or immunoprecipitation as described below. Samples were electrophoresed on 10% SDS-polyacrylamide gels and electro-transferred to nitrocellulose membranes (Hybond ECL, Amersham Biosciences). Membranes were subjected to immunoblotting with anti-T7 tag antibody (Novagen), anti-Xpress (Invitrogen), anti-JNK (D-2, Santa Cruz Biotechnology), anti-M3/6 polyclonal (also used to detect GST) (5), or anti-MKP7 polyclonal and horseradish peroxidase-conjugated secondary antibodies (SeraLab). Blots were developed using ECL reagents (Amersham Biosciences). For immunoprecipitation or pull down experiments, extracts were made as above and incubated with glutathione-Sepharose 4B (Amersham Biosciences) for GST-containing constructs, or with 1 µl of anti-MKP7 polyclonal antiserum or anti-T7 tag antibody with protein G-Sepharose beads (Sigma) for 3 h. Beads were washed three times in lysis buffer and resuspended in an appropriate volume of gel loading buffer. ND7 and N1E-115 cells were lysed in Triton lysis buffer (TLB) containing 20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 25 mM beta -glycerophosphate, 2 mM sodium pyrrophosphate, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin. Lysis was performed on ice for 30 min and following centrifugation (14,000 × g for 20 min), cleared lysates were incubated with anti-MKP7 or anti-M3/6 sera and protein G- or A-Sepharose beads, respectively, overnight at 4 °C. Washing, SDS-PAGE, and immunoblotting were performed as described above, and the blots were probed with anti-JIP-1 monoclonal antibody (22). COS-7 cells were harvested in TLB, and following SDS-PAGE and electro-transfer to nitrocellulose membranes, blots were probed with anti-HA (Santa Cruz Biotechnology), T7 tag, or anti-phospho-JNK (Cell Signaling Technology) antibodies. For experiments to determine c-Jun phosphorylation levels, COS-7 cells were lysed directly into gel loading buffer, and blots probed with anti-phospho-c-Jun (Ser-73) antibody (Cell Signaling Technology).

Immunofluorescence-- N1E-115 cells were grown on glass coverslips coated with poly-L-ornithine (Sigma). Cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde (Sigma), and lysed in 0.2% Triton X-100/phosphate-buffered saline. Indirect immunofluorescence was performed by incubation with monoclonal JIP-1 or polyclonal MKP7 antibodies in 3% bovine serum albumin/phosphate-buffered saline overnight at 4 °C. The appropriate fluorescein isothiocyanate-conjugated and Texas-Red-conjugated secondary antibodies were used (Jasckson Immunoresearch). Nuclei were detected with DAPI (Molecular Probes). Images were prepared using a Zeiss Axioplan-2 conventional fluorescence microscope.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Selective Binding of JIP-1 and JIP-2 to the Dual-specificity Phosphatases MKP7 and M3/6-- Sub-populations of JNK bind to scaffold proteins, therefore we asked whether distinct phosphatases regulate these distinct pools of JNK. MKP7 and M3/6 are DSPs that preferentially target JNK compared with other MAPKs (7, 10-12). We examined whether these DSPs could associate with the JIP family of scaffold proteins to regulate JNK activity. We co-expressed epitope-tagged JIP-1, JIP-2, and JIP-3 with either MKP7 or M3/6 and examined JIP precipitates for the presence of the DSPs using appropriate polyclonal antibodies. JIP-1 and JIP-2, but not JIP-3, associated with MKP7 (Fig. 1A, compare lanes 6, 9, and 12) and M3/6 (Fig. 1B, compare lanes 2, 4, and 6), suggesting that these phosphatases interact with a specific subset of JNK scaffold proteins. To determine whether the JIP proteins selectively interacted with MKP7 and M3/6 compared with other DSPs, we co-expressed GST-tagged JIP-1 and epitope-tagged versions of the DSPs in cells. Co-precipitation analysis demonstrated that MKP7 was detected in JIP-1 precipitates (Fig. 1C, lane 8). In contrast we detected no interaction with the DSPs MKP2, MKP4, and PAC1 (Fig. 1C, lanes 2, 4, and 6) or with MKP1 and MKP5.2 Our data therefore show that JIP-1 binds to a restricted set of DSPs including MKP7 and M3/6.


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Fig. 1.   Selective binding of JIP scaffold proteins to the dual-specificity phosphatases MKP7 and M3/6. A, plasmids expressing T7-tagged JIP-1, JIP-2, and JIP-3 (0.2 µg each) were introduced into 293T cells together with a plasmid expressing MKP7 (0.75 µg). In the control experiment (lanes 1-3) 0.2 µg of the parental vector pCDNA3 was introduced with MKP7. JIP-containing complexes were isolated by immunoprecipitation with anti-T7 tag antibody from 5 × 106 cells. The precipitates (T7-IP) were examined by immunoblot for the presence of MKP7 using a polyclonal MKP7-specific antiserum. The presence of JIP proteins and MKP7 in the original extract (Total extract) and the supernatant following immunoprecipitation (Supernatant) are also shown (0.2 × 106 cell equivalents loaded on gel). B, T7-tagged JIP-1, JIP-2, and JIP-3 (0.2 µg) were expressed in 293T cells with M3/6 (0.75 µg). For control experiments the parent vector pCDNA3 was co-transfected with the plasmid expressing M3/6. JIP-containing complexes were isolated by immunoprecipitation with anti-T7 tag antibody from 5 × 106 cells. The precipitates (T7-IP) were examined by immunoblot for the presence of M3/6 using a polyclonal antiserum specific for M3/6. The relative expression levels of M3/6 and T7-JIP proteins are also depicted (0.2 × 106 cell equivalents loaded on gel). C, plasmids expressing GST or GST-JIP-1 (0.2 µg) were co-transfected into 293T cells with plasmids expressing the XPress-tagged DSPs MKP4, MKP2, PAC1, and MKP7 (0.75 µg). JIP-1-containing complexes were isolated with glutathione-Sepharose beads (GST Pull down) from 5 × 106 cells, and the presence of the phosphatases in the JIP-1 precipitates was examined by immunoblot using Xpress tag antibody. The relative expression levels of GST, GST-JIP-1, and the DSPs are also shown (0.2 × 106 cell equivalents loaded on gels).

In Vivo Association of JIP-1 with MKP7 and M3/6-- JIP-1 is strongly expressed in neuronal cells (26, 29, 32), whereas MKP7 is reported to be abundantly expressed in brain (11). Using appropriate antibodies, we sought to establish whether endogenous complexes of JIP-1 and MKP7 or M3/6 are present in neuronal cells. Antisera against M3/6 (5) and a JIP-1 monoclonal antibody (22) have been described previously. We generated polyclonal antisera against MKP7, which recognized exogenously expressed T7-tagged MKP7 on immunoblots of total COS-7 cell extracts (Fig. 2A) and recognized an endogenous protein corresponding to the size of MKP7 in extracts of the neuronal cell line N1E-115 (Fig. 2B). The MKP7 antiserum also immunoprecipitated the T7-tagged MKP7 from COS-7 extracts (Fig. 2A), indicating its suitability for co-immunoprecipitation experiments.


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Fig. 2.   JIP-1 associates with MKP7 and M3/6 in vivo. A, total cell extracts prepared from COS-7 cells expressing T7-tagged MKP7 (0.75 µg of plasmid transfected) or from untransfected cells were examined by immunoblot using polyclonal MKP7 antisera (0.2 × 106 cell equivalents loaded on gel) (left hand panel). Extract from 5 × 106 cells was subjected to immunoprecipitation using the MKP7 antisera (MKP7 IP), and the presence of T7-MKP7 in the precipitates was examined by immunoblot using the T7 tag antibody (right hand panel). The asterisk denotes the position of the immunoglobulin heavy chain component of the MKP7 antibody used for immunoprecipitation. The positions of size markers are indicated (in kDa). B, immunoblot using MKP7 antisera of total extract from N1E-115 cells (50 µg loaded on gel). The positions of size markers are indicated (in kDa). C, extracts of ND7 cells (0.5 mg) were subject to immunoprecipitation (IP) with polyclonal MKP7 antisera. The presence of JIP-1 in the precipitates was examined by immunoblot with a monoclonal JIP-1 antibody. A control IP was performed using rat immunoglobulin. For comparison, 20 µg of the original extract (Total Extract) was loaded on the gel. D, extracts of N1E-115 cells (0.5 mg) were subjected to immunoprecipitation with a rabbit anti-M3/6 polyclonal antiserum. The presence of JIP-1 in the precipitates was examined by immunoblot with a monoclonal JIP-1 antibody. For comparison, 20 µg of the original extract (Total Extract) was loaded on the gel. E, detection of endogenous JIP-1 (green) and MKP7 (red) expression in N1E-115 cells by immunofluorescence analysis. The nucleus was detected by staining DNA with DAPI (blue). F, double-label immunofluorescence analysis of JIP-1 (green) and MKP7 (red) in N1E-115 cells. The images were overlaid (Merge) to demonstrate co-localization (yellow). The nucleus was detected by staining DNA with DAPI (blue).

We performed co-immunoprecipitation analysis using the MKP7 and M3/6 antisera, and we detected endogenous JIP-1 in both MKP7 and M3/6 precipitates from extracts of the neuronal cell lines ND7 and N1E-115, respectively (Fig. 2, C and D). These data indicate that endogenously expressed JIP-1 can associate with MKP7 and M3/6. Previously, JIP-1 has been demonstrated to reside in the cytoplasm of N1E-115 cells and to be concentrated at the tips of the neurites (29). Both exogenously expressed and endogenous MKP7 have been reported to be cytoplasmic (10-12), although there is evidence that it is a nuclear shuttle protein (11). We therefore investigated whether JIP-1 and MKP7 co-localized in N1E-115 cells. As shown in Fig. 2E, JIP-1 immunostaining is present in the cell bodies and is also concentrated at the tips of neurites as previously described (29). MKP7 staining is present in the cell bodies but does not appear to be concentrated at the tips of the neurites (Fig. 2E). We did not detect JIP-1 or MKP7 immunostaining in the nuclei (Fig. 2E). Dual-labeling experiments indicate that JIP-1 and MKP-7 co-localize in the cell bodies, but only JIP-1 staining is present in the neurite tips (Fig. 2F). The patterns of JIP-1 and MKP7 localization in N1E-115 cells are therefore distinct but overlapping. Taken together with the immunoprecipitation data, the results suggest that only a proportion of JIP-1 is in a complex with MKP7 and M3/6 in resting neuronal cells.

MKP7 Binds to JIP-1 Independently of JNK Binding-- It has previously been demonstrated that immunoprecipitates of over-expressed MKP7 (10, 11) and M3/6 (5) contain JNK and that JIP-1 can directly bind to JNK (19, 21). The formation of complexes between JIP-1 and MKP7 or M3/6 might therefore be mediated by an interaction of the phosphatase with JNK. To examine this question, co-immunoprecipitation analysis was used to determine the ability of MKP7 and M3/6 to bind to a JIP-1 mutant with a deletion in the JNK binding domain (JIP-1 Delta JBD) (Fig. 3, A and B). MKP7, but not JNK, bound to JIP-1 Delta JBD, indicating that MKP7 can bind to JIP-1 independently of JNK (Fig. 3B, lane 3). Similar results were obtained for M3/6 binding to JIP-1 Delta JBD.2 We further demonstrated that MKP7 could not bind to the N-terminal region of JIP-1 that includes the JNK binding domain (residues 1-282) but did bind to a C-terminal fragment of JIP-1 (residues 282-707) that does not associate with JNK (Fig. 3B, lanes 4 and 5). Very weak binding of a shorter C-terminal fragment of JIP-1 (amino acids 471-707) to MKP7 was also detected (Fig. 3B, lane 6). These results indicate that C-terminal regions of JIP-1 are required for binding MKP7 and that MKP7 forms a stable interaction with JIP-1 in the absence of JNK binding. However, it does not rule out the possibility that interactions between MKP7 and JNK may help to stabilize MKP7 binding to JIP-1.


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Fig. 3.   MKP7 binds to JIP-1 independently of JNK binding. A, schematic of the JIP-1 deletion constructs used in the experiments. Numbers refer to amino acid positions. JBD, JNK binding domain; PTB, phosphotyrosine binding domain; SH3, Src homology-3 domain. B, constructs expressing GST or GST-tagged JIP-1 deletion mutants (0.2 µg) were introduced into 293T cells together with expression vectors for either MKP7 or JNK3 (0.75 µg). JIP-1-containing complexes were isolated from extracts of 5 × 106 cells with glutathione-Sepharose beads (GST Pull down). The presence of MKP7 and JNK3 in the extracts and precipitates was examined by immunoblot with appropriate antibodies (0.2 × 106 cell equivalents loaded on gels). C, schematic of the MKP7 deletion constructs used in the experiments. Numbers refer to amino acid positions. DSP, dual-specificity phosphatase domain; NLS, nuclear localization sequence; NES, nuclear export sequence. D, constructs expressing GST or GST-JIP-1 were introduced into 293T cells with constructs expressing full-length XPress-tagged MKP7 or MKP7 deletions. JIP-1-containing complexes were isolated from extracts of 5 × 106 cells with glutathione-Sepharose beads (GST Pull down), and the presence of MKP7 deletions was examined by immunoblotting with XPress tag antibody. The expression levels of GST, GST-JIP-1, and MKP7 deletions in the extracts are also shown (0.2 × 106 cell equivalents loaded on gels).

We next sought to identify the region of MKP7 that mediates its binding to JIP-1. We performed co-precipitation analysis of MKP7 deletion mutants with JIP-1 (Fig. 3, C and D). An MKP7 deletion mutant containing amino acids 1-394 did not bind to JIP-1, whereas a mutant containing amino acids 1-443 did bind JIP-1, albeit more weakly than full-length MKP7 (Fig. 3D, compare lanes 1, 5, and 7). These experiments identify the sequence between amino acids 394 and 443 as important for binding. There was increased binding to JIP-1 of an MKP7 mutant containing amino acids 1-552 (Fig. 3D, lane 11), indicating that additional sequences within the C terminus also contribute to binding. The C terminus of MKP7 is homologous to that of M3/6 but not to other DSPs, which probably explains the selective binding of MKP7 and M3/6 by JIP-1. The binding of JIP-1 to the C-terminal region of MKP7 is distinct from the JNK-binding region located in the N terminus of MKP7 (10), providing additional evidence that MKP7 binding to JIP-1 is independent of JNK binding. Interestingly, an MKP7 mutant lacking the extended C terminus, and therefore unable to bind JIP-1, is significantly more active against JNK in co-expression studies than wild-type MKP7.2 This suggests that the C terminus of MKP7 may perform a regulatory function, and this in part may be mediated by binding to regulatory proteins such as JIP-1.

MKP7 Blocks JIP-1 Enhanced Activation of JNK and c-Jun Phosphorylation-- JIP-1 binds to JNK, MKK7, and MLKs resulting in enhanced activation of JNK (21). We therefore tested the possibility that the interaction of MKP7 with JIP-1 would enhance the ability of this phosphatase to dephosphorylate the pool of JNK associated with the JIP-1 scaffold. As previously demonstrated, co-expression of full-length JIP-1 with MLK3 and JNK significantly enhanced the phosphorylation of JNK by MLK3 (Fig. 4A, compare lanes 5 and 8). The expression of MKP7 at a level that did not block JNK activation by MLK3 in the absence of JIP-1 was nevertheless able to completely block the ability of JIP-1 to enhance JNK activation by MLK3 (Fig. 4A, compare lanes 6 and 9). This effect of MKP-7 depended upon its phosphatase activity. A catalytically inactive mutant of MKP7 (MKP7 C/S) that still binds JIP-1 did not block the JIP-1-mediated enhancement of JNK activation (Fig. 4A, lane 10). These data demonstrate that the ability of MKP7 to dephosphorylate JNK is greatly enhanced by the JIP-1 scaffold protein.


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Fig. 4.   MKP7 blocks JIP-1-enhanced JNK activation and c-Jun phosphorylation. A, constructs expressing HA-tagged JNK2 (0.1 µg) together with HA-MLK3 (0.1 µg), T7-JIP-1 (0.2 µg), T7-MKP7, or T7-MKP7 (C/S) (0.1 µg) were introduced into COS-7 cells as indicated. The presence of phospho-JNK in the cell extracts (0.2 × 106 cell equivalents loaded on gel) was examined by immunoblot analysis using an antibody specific for the phosphorylated form of JNK. The presence of total JNK2, MLK3, and JIP-1 in the cell extracts (0.2 × 106 cell equivalents loaded on gels) was examined by immunoblotting with anti-HA and T7 tag antibodies, respectively. Expression levels of MKP7 were detected by immunoprecipitation from extracts (3 × 106 cells) using anti-MKP7 polyclonal antibody and blotting the immunoprecipitates with T7 tag antibody. The experiment was repeated three times with similar results. B, constructs expressing HA-tagged c-Jun (0.1 µg) together with HA-JNK2 (0.1 µg), HA-MLK3 (0.1 µg), T7-JIP-1 (0.2 µg), and T7-MKP7 (0.2 µg) were introduced into COS-7 cells as indicated. The presence of phospho-c-Jun in the cell extracts (0.2 × 106 cell equivalents loaded on gel) was examined by immunoblot using an antibody specific for the phosphorylated form of c-Jun. The presence of total c-Jun, JNK, MLK3, and JIP-1 in the cell extracts (0.2 × 106 cell equivalents loaded on gels) was examined by immunoblotting with anti-HA and T7 tag antibodies. The experiment was repeated twice with similar results.

One of the major targets of the JNK-signaling pathway is the transcription factor c-Jun (1). JNK phosphorylates c-Jun at Ser-63 and Ser-73 leading to an increase in its transcriptional activity (1). Previous genetic studies (32) have indicated that JIP-1-signaling complexes are involved in transducing JNK signaling to the c-Jun transcription factor. We therefore determined the effect of MKP7 on the ability of JIP-1 to enhance JNK-mediated c-Jun phosphorylation. Co-expression of JIP-1 with MLK3, JNK, and c-Jun significantly enhanced c-Jun phosphorylation (Fig. 4B, compare lanes 3 and 5). The expression of MKP7 impairs the ability of JIP-1 to enhance JNK-mediated c-Jun phosphorylation (Fig. 4B, compare lanes 5 and 6). Taken together, these results provide evidence that MKP7 can be recruited to the JIP-1 complex to down-regulate JNK signaling, leading to reduced phosphorylation of the JNK target c-Jun.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A number of scaffold proteins that regulate the activities of MAPK pathways have recently been characterized (17, 18). Our data provides the first evidence that MAPK scaffold proteins can recruit protein phosphatases to modulate MAPK activity. The JIP group of scaffold proteins regulates the activity of the JNK MAPK-signaling pathway by binding to the MLK-MKK7-JNK-signaling module (21-23). We have demonstrated that the JIP family proteins JIP-1 and JIP-2 also selectively associate with the DSPs M3/6 and MKP-7 (Figs. 1-3), and this can lead to the dephosphorylation and inactivation of JNK and reduced phosphorylation of c-Jun (Fig. 4).

Our results suggest a model whereby MKP7 and M3/6 are recruited to JIP scaffold proteins to dephosphorylate and inactivate JNK. However, the precise mechanism by which this occurs is unclear. One possibility is that these DSPs act to continually suppress the activation of JNK through the JIP-1 scaffold protein. This raises the possibility that inhibition of phosphatase action, either by inhibition of enzymatic activity or disruption of the interaction of the phosphatase with JIP-1, could contribute to the activation of JNK by JIP-1. Alternatively, phosphatase activity could be required at some specific point in the cycle of JIP-1 function. For example, the phosphatase may act to suppress inappropriate JNK activation, either by particular stimuli or at particular cell locations. The fact that in resting neuronal cells only a fraction of endogenous JIP-1 forms a stable complex with MKP7 and M3/6 (Fig. 2, C and D) suggests that the recruitment of the DSPs may not be a general mechanism for keeping JIP-mediated JNK signaling repressed in the absence of signal. This is supported by co-localization studies that demonstrate that the patterns of JIP-1 and MKP7 immunostaining in N1E-115 cells are overlapping but also distinct (Fig. 2, E and F). It is possible therefore that DSPs are recruited to JIP proteins at a particular cell location (e.g. in the cytoplasm of the cell body) or following stimulation of cells. Our preliminary experiments demonstrate that JIP-1 binding to MKP7 is not affected by oxidative stress,2 but future studies will be aimed at determining whether this interaction can be regulated either positively or negatively by other signals.

In the future it will be of interest to determine whether other regulators of MAPK pathways recruit specific protein phosphatases to down-regulate these pathways. It seems likely that the targeting of DSPs to specific JNK-signaling modules may be a common theme. Recently a novel DSP, SAPK pathway-regulating phosphatase 1, was described, which when over-expressed can bind to MKK7 and apoptosis signal-regulating kinase 1, potentially acting as a type of scaffold protein itself (35, 36). Expression of SAPK pathway-regulating phosphatase 1 specifically blocked JNK activation by tumor necrosis factor-alpha (tumor necrosis factor-alpha ), indicating that the formation of this complex may have a distinct function in cells (35, 36).

Although the binding of protein phosphatases to regulators of MAPK-signaling pathways is a novel finding, there are precedents from other signaling systems for the recruitment of protein kinases and protein phosphatases to the same regulatory protein. For example, some members of the protein kinase A anchoring protein (AKAP) family of cAMP-dependent protein kinase-binding proteins also associate with protein phosphatases, thereby co-localizing both positive and negative regulators of a common substrate at distinct locations in cells (37). Such recruitment of positive and negative regulators of signaling pathways by scaffolding or anchoring proteins may therefore be a common mechanism for achieving signaling specificity in cells and permitting the rapid modulation of the activities of target proteins by phosphorylation and dephosphorylation.

    ACKNOWLEDGEMENTS

We thank R. J. Davis, M. Karin, A. Ashworth, and J. Downward for reagents.

    FOOTNOTES

* This work was supported by the Association for International Cancer Research, the Wellcome Trust, and Cancer Research UK.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this study.

|| A Lister Institute-Jenner Research Fellow.

** To whom correspondence should be addressed. Tel.: 44-161-275-7825; Fax: 44-161-275-5082; E-mail: alan.j.whitmarsh@man.ac.uk.

Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M207324200

2 E. A. Willoughby, G. R. Perkins, M. K. Collins, and A. J. Whitmarsh, unpublished results.

    ABBREVIATIONS

The abbreviations used are: JNK, c-Jun N-terminal kinase; JIP, JNK-interacting protein; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; MKKK, MAPKK kinase; MKP, MAPK phosphatase; MLK, mixed lineage kinase; ERK2, extracellular signal regulated kinase 2; JBD, JNK binding domain; GST, glutathione S-transferase; DSP, dual-specificity phosphatase; HA, hemagglutinin; DAPI, 4',6-diamidino-2-phenylindole.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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