From the 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
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ABSTRACT |
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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.
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 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
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.
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.
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.
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
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.
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.
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- 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin-2 (19-26). JIP-1 and JIP-2 share extensive
sequence homology and are mainly expressed in neuronal tissues, testis,
and
-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),
-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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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).
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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).
JBD) (Fig.
3, A and B). MKP7,
but not JNK, bound to JIP-1
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
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).
View larger version (44K):
[in a new window]
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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(tumor necrosis factor-
), indicating that the formation of this complex may have a distinct function in cells (35, 36).
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ACKNOWLEDGEMENTS |
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We thank R. J. Davis, M. Karin, A. Ashworth, and J. Downward for reagents.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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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.
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