From the Medical Research Council Laboratory of
Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom, the
¶ Medical Research Council Protein Phosphorylation Unit,
Department of Biochemistry, University of Dundee, MSI/WTB Complex,
Dundee DD1 5EH, United Kingdom, and the
Department of Neurology,
E.D. Adrian Building, University of Cambridge, Robinson Way,
Cambridge CB2 2PY, United Kingdom
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ABSTRACT |
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Mechanisms for selective targeting to unique
subcellular sites play an important role in determining the substrate
specificities of protein kinases. Here we show that stress-activated
protein kinase-3 (SAPK3, also called ERK6 and p38 Stress-activated protein kinases
(SAPKs)1 are
mitogen-activated protein kinase (MAPK) family members that are
activated by cellular stresses, bacterial lipopolysaccharide, and the
cytokines interleukin-1 and tumor necrosis factor (reviewed in Ref. 1). A major challenge in this field is to identify the physiological substrates and functions of each SAPK. SAPK1 (or c-Jun amino-terminal kinase) consists of a number of closely related isoforms that phosphorylate Ser63 and Ser73 in the activation
domain of c-Jun, thereby increasing its transcriptional activity
(2-4). The same sites in c-Jun also become phosphorylated when cells
are exposed to the stresses and cytokines that activate SAPK1 (2,
4-6), suggesting that c-Jun is a physiological substrate for SAPK1. A
second class of SAPK comprises SAPK2a (also called p38/RK/CSBPs) (7-9)
and SAPK2b (10) (also called p38 A third class of SAPK consists of the more recently identified SAPK3
(also called ERK6 and p38 Materials--
Full-length human Yeast Two-hybrid System Screening--
Yeast two-hybrid
screening (27) was performed using an adult human brain expression
library (CLONTECH) containing cDNAs fused to
the GAL4 transactivation domain of pACT2 and rat SAPK3 DNA (20)
subcloned into vector pAS2-1, which contains the GAL4 DNA binding
domain. The plasmids were transformed into Y190 yeast cells, and
positive clones were selected on triple minus plates (Leu ELISA--
GST fusion proteins of PDZ domain-containing proteins
were bound to 96-well Micro Test plates (Falcon) at 10 µg/ml in 50 mM Tris-HCl (pH 7.9). Plates were incubated overnight at
4 °C, washed three times in phosphate-buffered saline (PBS) and
blocked with 1% bovine serum albumin in PBS for 1 h at 37 °C.
After washing four times in PBS, serial 1:3 dilutions (starting at 200 µg/ml) of thioredoxin-SAPK3(1-367) or thioredoxin-SAPK3(1-363) in
1% bovine serum albumin/PBS + 0.1% Tween 20 (w/v) were added and allowed to bind for 1 h at 37 °C. Plates were washed four times in PBS + 0.1% Tween 20, incubated with anti-thioredoxin antibody (1:3,000, Invitrogen) for 1 h at 37 °C, washed four times in
PBS + 0.1% Tween 20, and incubated with goat anti-mouse IgG-conjugated peroxidase (1:2,000, Bio-Rad) for 1 h at 37 °C. Plates were
washed three times in PBS, followed by the addition of 100 µl of 50 mM citrate-phosphate buffer (pH 5.0) + 0.5 mg/ml
o-phenylenediamine (Sigma). After 5 min the color reaction
was stopped by addition of 20 µl of 8 N
H2SO4 and absorbance at 450 or 490 nm
determined using a microplate reader (Molecular Devices).
Identification of Phosphorylation Sites--
GST- Immunofluorescence--
Pectoral and semitendinous muscles were
dissected from five adult Sprague-Dawley rats and kept at Transfection and Immunoprecipitation--
Full-length rat SAPK3
and human To identify SAPK3 substrates, we performed a yeast two-hybrid
screen of a human brain cDNA library using rat SAPK3 as bait. This
screen yielded two independent clones encoding residues 85-505 of
), a member of the
mitogen-activated protein kinase family that is abundantly expressed in
skeletal muscle, binds through its carboxyl-terminal sequence
-KETXL to the PDZ domain of
1-syntrophin. SAPK3
phosphorylates
1-syntrophin at serine residues 193 and 201 in
vitro and phosphorylation is dependent on binding to the PDZ
domain of
1-syntrophin. In skeletal muscle SAPK3 and
1-syntrophin
co-localize at the neuromuscular junction, and both proteins can be
co-immunoprecipitated from transfected COS cell lysates.
Phosphorylation of a PDZ domain-containing protein by an associated
protein kinase is a novel mechanism for determining both the
localization and the substrate specificity of a protein kinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2; Ref. 11), whose substrates
include other protein kinases, such as MAPK-activated protein kinases-2
and -3 (8, 12), MAPK-interacting protein kinases-1 and -2 (13, 14),
p38-regulated/activated protein kinase (15), and mitogen- and
stress-activated protein kinases-1 and -2 (16), as well as several
transcription factors (1). Identification of physiological substrates
of SAPK2a (p38) and SAPK2b (p38
2) is greatly facilitated because of
the specific inhibition of these enzymes by the cell-permeant pyridinyl
imidazole SB 203580 and related compounds (9, 17-19).
) (20-23) and SAPK4 (also called p38
)
(10, 11, 24, 25). The mRNAs encoding these enzymes are present in
all mammalian tissues examined, with the mRNA encoding SAPK3 being
most abundant in skeletal muscle (20-22). SAPK3 and SAPK4 are not
inhibited by SB 203580 (10, 23), and consequently only little is known
about their substrates. The transcription factor ATF2 is a good
substrate of SAPK3 in vitro (23), whereas stathmin has been
proposed as a physiological substrate of SAPK4 (26). Here we identify
1-syntrophin as a substrate for SAPK3 and show that phosphorylation
is dependent on the interaction of the carboxyl-terminal sequence
-KETXL of SAPK3 with the PDZ domain of
1-syntrophin. In
skeletal muscle SAPK3 and
1-syntrophin were found to co-localize at
the neuromuscular junction and throughout the sarcolemma.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-syntrophin was obtained by
polymerase chain reaction from human skeletal muscle cDNA. It was
subcloned into pACT2 (Stratagene) for yeast two-hybrid screening or
pGEX4T-1 (Amersham Pharmacia Biotech) for bacterial expression as a GST fusion protein.
1-Syntrophin (78-179) and
1-syntrophin
(174-505) were produced by polymerase chain reaction, as were human
1-syntrophin (103-204) and human neuronal nitric-oxide synthase
(9-108). Expression and activation of rat GST-SAPK3 have been
described (23). Rat SAPK3(1-363) was produced by polymerase chain
reaction and subcloned into pGEX4T-1 for expression as GST fusion
protein. For some experiments SAPK3(1-363) and SAPK3(1-367) were
subcloned into the yeast two-hybrid vector pAS2-1 (Stratagene) or the
thioredoxin fusion protein vector pET32a (Novagen). Site-directed
mutagenesis was used to produce L367VSAPK3, followed by subcloning into
pGEX4T-1 and expression as a GST fusion protein. All constructs were
verified by DNA sequencing. Expression and activation of recombinant
MAPK, SAPK2a, SAPK2b, and SAPK4 have been described (10).
, Trp
, His
) + 25 mM 3-aminotriazole and assayed for
-galactosidase
activity. Two million clones were screened, and two positives were
obtained. Positive clones were co-transformed with either the bait
vector or the original pAS2-1 (used as a control) into yeast to
confirm the interaction. All the constructs that were used in other
interaction experiments were from polymerase chain reaction products
subcloned into pAS2-1 or pACT2 and were confirmed by DNA sequencing.
1-syntrophin
(0.5 µM) was incubated at 30 °C for 1 h with
activated GST-SAPK3 (2 units/ml) (23), 10 mM magnesium
acetate, and 100 µM [
-32P]ATP in a total
volume of 200 µl of 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.1 mM sodium orthovanadate, and 0.1%
(v/v) 2-mercaptoethanol. After SDS-polyacrylamide gel electrophoresis
and autoradiography, the band corresponding to 32P-labeled
1-syntrophin was excised and digested with trypsin, and the
phosphopeptides generated were chromatographed on a Vydac 218TP54
C18 column equilibrated with 0.1% (v/v) trifluoroacetic acid, and the column was developed with a linear acetonitrile gradient.
The flow rate was 0.8 ml/min, and fractions of 0.4 ml were collected.
The two peaks of 32P radioactivity were analyzed by solid
and gas phase sequencing (28) and also by electrospray mass
spectrometry to identify the peptide sequences and sites of
phosphorylation. SAPK3 was assayed routinely with MBP as substrate
(23). Phosphorylation of
1-syntrophin by wild-type GST-SAPK3,
GST-SAPK3(1-363), and GST-L367VSAPK3 was carried out in the same
manner. Reactions were stopped by the addition of 1 ml of 10% (w/v)
trichloroacetic acid, and after centrifugation for 10 min at
13,000 × g, the supernatants were discarded. The
pellets were washed three times with 1 ml of 25% (w/v) trichloroacetic
acid, and 32P incorporation was measured by Cerenkov
counting. Incorporation of phosphate into substrate was kept below 0.1 mol phosphate/mol substrate in all experiments to ensure that initial
rate conditions were met.
70 °C
until use. Cryosections (10 µm) were dipped in acetone, air-dried,
and fixed in 2% paraformaldehyde (w/v). Following a 5-min wash in PBS,
sections were incubated overnight at 4 °C in 10
7
M tetramethylrhodamine
-bungarotoxin (Molecular Probes,
Inc.) diluted in PBS. Tissue sections were then washed for 15 min in PBS and fixed for 5 min in ethanol. For double staining, tissue sections were further incubated overnight with anti-SAPK3 serum R5
(diluted 1:200). R5 was raised in a rabbit against the synthetic peptide KPPRQLGARVPKETAL (corresponding to residues 352-367 of rat
SAPK3) conjugated to keyhole limpet hemocyanin. After a 30-min wash in
PBS, tissue sections were incubated for 2 h at room temperature with biotinylated anti-rabbit secondary antibody (diluted 1:200, Vector
Laboratories), and, following a further 30-min wash in PBS, they were
incubated for 1 h at room temperature with fluorescein-avidin D
(diluted 1:200, Vector Laboratories). Sections that were triple stained
were washed in PBS, blocked using the Vector blocking kit, and
incubated overnight at 4 °C with anti-
1-syntrophin serum SYN17
(diluted 1:50) (29). Incubation with biotinylated secondary antibody
and washings were done as for double staining and sections were then
incubated for 1 h at room temperature with
7-amino-4-methylcoumarin-3-acetic acid-streptavidin (diluted 1:50,
Boehringer Mannheim). Sections were mounted using Vectashield mounting
medium. Immunofluorescence was observed using a Leitz DMRD fluorescence
microscope using filters for rhodamine, fluorescein, and
7-amino-4-methylcoumarin-3-acetic acid. In parallel experiments, muscle
sections were single stained with tetramethylrhodamine
-bungarotoxin, antiserum R5, or antiserum SYN17. As a control for
the specificity of staining, diluted antiserum R5 was incubated with 10 µM recombinant GST-SAPK3 prior to staining. Moreover, in
double or triple stainings, tetramethylrhodamine
-bungarotoxin and
antibodies R5 or SYN17 were alternatively omitted.
1-syntrophin cDNAs were subcloned into the eukaryotic
expression vector pSG5 and COS cells transiently transfected with 10 µg/ml plasmid DNA using DEAE-dextran chloroquine. After 48 h
cells transfected with SAPK3 alone and double transfected with SAPK3
and
1-syntrophin were lysed in 300 µl of buffer (20 mM
Tris acetate, pH 7.5, 0.27 M sucrose, 1 mM
EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM
-glycerophosphate, 0.1% 2-mercaptoethanol (v/v), 1 mM
benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin). Aliquots (100 µl) of cell lysates were incubated
for 90 min at 4 °C on a shaking platform with 20 µl of protein
A-Sepharose conjugated to 10 µl of anti-
1-syntrophin serum TROPHA.
TROPHA was raised in a rabbit against the synthetic peptide
ASGRRAPRTGLLELRAG (corresponding to residues 2-17 of human
1-syntrophin) coupled to keyhole limpet hemocyanin. The suspensions were centrifuged for 1 min at 13,000 rpm, and the immunoprecipitates were washed twice with 1 ml of lysis buffer containing 0.5 M NaCl and once with 1 ml lysis buffer, followed by
resuspension in gel loading buffer. Immunoprecipitates were detected
with anti-
1-syntrophin serum TROPHA and anti-SAPK3 serum R5.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-syntrophin.
1-Syntrophin is a peripheral membrane protein that
comprises two pleckstrin homology domains, a PDZ domain and a unique
carboxyl-terminal domain, with the PDZ domain being inserted into the
first pleckstrin homology domain (30-33). The related proteins
1-syntrophin and
2-syntrophin share a similar domain organization (31-34). Syntrophins are believed to function as modular adapters that recruit signaling proteins to the dystrophin-glycoprotein complex at the plasma membrane (35). The yeast two-hybrid system was
used to examine the domains that are responsible for the
1-syntrophin-SAPK3 interaction (Fig.
1). Full-length
1-syntrophin
interacted with SAPK3. The shortest construct that was positive when
paired with SAPK3 contained the PDZ domain (residues 78-179) of
1-syntrophin. By contrast, a construct extending from the end of the
PDZ domain to the carboxyl terminus of
1-syntrophin (residues
174-505) failed to interact with SAPK3, establishing that the PDZ
domain of
1-syntrophin mediates the binding to SAPK3. PDZ domains
are known to interact with the carboxyl termini of proteins that have
the consensus sequence -E(S/T)XV (36, 37). The carboxyl
terminus of rat SAPK3 (amino acid sequence -ETAL) (20) is similar to
this consensus sequence. Deletion of the last four amino acids of SAPK3
prevented its association with
1-syntrophin, demonstrating that this
sequence is necessary for the interaction (Fig. 1). The syntrophin
constructs were also expressed as GST fusion proteins and their binding
to thioredoxin-SAPK3 assessed by ELISA (Fig. 1). As in the yeast two-hybrid system, SAPK3 bound through its carboxyl-terminal four amino
acids to the PDZ domain of
1-syntrophin. Similarly, SAPK3 interacted
with the PDZ domain of
1-syntrophin (Fig. 1), whereas it failed to
bind to the PDZ domain of neuronal nitric-oxide synthase (Fig. 1),
which forms homotypic interactions with the PDZ domain of
1-syntrophin and PDZ domains 1 and 2 of postsynaptic density protein
95 (PSD95/SAP90) (38). The PDZ domain of neuronal nitric-oxide synthase
bound to
1-syntrophin both in the yeast two-hybrid system and as
judged by ELISA (not shown).
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Fig. 1.
Interactions between SAPK3 and
1-syntrophin. A, interaction of
SAPK3 with the PDZ domain of
1-syntrophin. Binding of GAL4 fusion
constructs of human
1-syntrophin, the PDZ domain of human
1-syntrophin, and the PDZ domain of human neuronal nitric-oxide
synthase (nNOS) to rat SAPK3 was tested in the yeast
two-hybrid system. Interactions were measured by the activity of the
reporter genes HIS3 and
-galactosidase. HIS3 activity was judged by
growth in medium lacking histidine in the presence of 25 mM
3-aminotriazole and
-galactosidase activity was determined from the
time taken for the colonies to turn blue in
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside filter
lift assays performed at room temperature: +, 90-240 min;
, no
significant
-galactosidase activity. In vitro binding of
SAPK3 to the PDZ domain-containing proteins was tested by ELISA. The
two SAPK3-interacting clones isolated in the yeast two-hybrid screen
(shown as pACT2) encoded residues 85-505 of human
1-syntrophin.
B, interaction of human
1-syntrophin with full-length rat
SAPK3(1-367) but not with SAPK3(1-363).
Human 1-syntrophin contains nine (S/T)P sites located outside the
PDZ domain that are potential sites of phosphorylation by SAPKs (30,
31). Activated GST-SAPK3 phosphorylated GST-
1-syntrophin to 2 mol
phosphate/mol protein in vitro, and two
32P-labeled tryptic peptides were identified that
corresponded to residues 198-207 and 178-197, respectively (Fig.
2A). Solid and gas phase
sequencing, as well as electrospray mass spectrometry were used to
identify the phosphorylated residues as serines 193 and 201, which are
located between the PDZ domain and the second half of the first
pleckstrin homology domain (Fig. 1A). Initial rates of
phosphorylation showed that relative to myelin basic protein
1-syntrophin is a good substrate for SAPK3 but not for other SAPKs
or for p42 MAPK (Table I). SAPK3
phosphorylated
1-syntrophin at approximately the same rate as it
phosphorylated MBP, its standard substrate (Table I). Phosphorylation
of
1-syntrophin by SAPK3 was dependent on the carboxyl-terminal four
amino acids of SAPK3, as demonstrated by the following three separate
lines of evidence (Fig. 2, B-D).
1-Syntrophin was a
poor substrate for GST-SAPK3(1-363), which lacks the carboxyl-terminal
four amino acids, whereas MBP was an equally good substrate for both
GST-SAPK3(1-363) and GST-SAPK3(1-367) (Fig. 2B).
Furthermore, preincubation of wild-type rat GST-SAPK3 with an antibody
raised against its carboxyl-terminal 16 amino acids prevented
phosphorylation of
1-syntrophin but not MBP (Fig. 2C).
Finally, preincubation of
1-syntrophin with synthetic peptides corresponding to the carboxyl-terminal 6 or 8 amino acids of rat SAPK3
prevented phosphorylation of
1-syntrophin by GST-SAPK3 (Fig.
2D).
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The carboxyl-terminal sequence -KETAL of mouse, rat, rabbit, and
zebrafish SAPK3 (20)2 or
-KETPL of human SAPK3 (10, 21, 22) is the most conserved sequence in
the carboxyl-terminal region of SAPK3 and differs from the prototypical
consensus PDZ domain-binding sequence (36, 37) by replacement of the
terminal valine with leucine. We therefore investigated the ability of
rat GST-L367VSAPK3 to bind and phosphorylate GST-1-syntrophin. By
ELISA, the binding of wild-type GST-SAPK3 to
1-syntrophin was
similar to that of mutant GST-L367VSAPK3 (Fig.
3A). The rate of
phosphorylation of
1-syntrophin by GST-L367VSAPK3 was slightly
faster than by wild-type GST-SAPK3 (Fig. 3B). However, both
mutant and wild-type SAPK3 phosphorylated
1-syntrophin to the same
extent (Fig. 3B). The phosphorylation of MBP by SAPK3 was
unaffected by the L367V mutation (Fig. 3C).
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If the association of SAPK3 with 1-syntrophin is physiologically
relevant, the two proteins should be co-localized in vivo. Both SAPK3 and
1-syntrophin are expressed at highest levels in skeletal muscle (20-22, 30, 31), where
1-syntrophin is associated with the sarcolemma and concentrated at the neuromuscular junction (39). We used immunofluorescence to examine the localization of SAPK3
in rat skeletal muscle. SAPK3 was found throughout the sarcolemma and
was concentrated at the neuromuscular junction, as indicated by its
co-localization with
-bungarotoxin, which visualizes nicotinic
acetylcholine receptors at the neuromuscular junction (Fig.
4). Moreover, double staining for SAPK3
and
1-syntrophin showed extensive co-localization, both at the
neuromuscular junction and throughout the sarcolemma (Fig. 4). The
staining was specific, because it was abolished by incubation of
diluted SAPK3 antiserum with 10 µM recombinant SAPK3 (not
shown). For an independent assessment of the
1-syntrophin-SAPK3
interaction, the ability of SAPK3 to co-immunoprecipitate with
1-syntrophin was examined in extracts from mammalian cells
co-transfected with both proteins.
1-Syntrophin and SAPK3 were
co-expressed transiently in COS cells. Immunoprecipitation was carried
out using an anti-
1-syntrophin antibody, and proteins present in the
pellet were immunoblotted using anti-
1-syntrophin and anti-SAPK3
antibodies. The strong signal seen for SAPK3 upon immunoprecipitation
with the anti-
1-syntrophin antibody indicates that
1-syntrophin
existed in a complex with SAPK3 in COS cell lysates (Fig.
5).
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DISCUSSION |
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SAPK3 is a protein kinase whose phosphorylation of 1-syntrophin
depends on the interaction between its carboxyl-terminal sequence and
the PDZ domain of this substrate. The carboxyl-terminal sequence of
SAPK3 thus provides a mechanism both for its selective targeting to
subcellular sites and for determining its substrate specificity. During
vulval induction in Caenorhabditis elegans, the PDZ
domain-containing protein LIN-7 is essential for localizing the
epidermal growth factor receptor-like tyrosine kinase LET-23 to cell
junctions by binding through its PDZ domain to the carboxyl-terminal sequence -KETCL of LET-23 (40-42). Similarly, protein kinase C
is
a protein kinase that is targeted to subcellular sites through the
interaction of its carboxyl-terminal sequence -QSAV with the PDZ domain
of the protein kinase C
-binding protein (PICK1) (43). Moreover, p70
S6 kinase has been shown to bind through its carboxyl-terminal sequence
to the PDZ domain of neurabin, suggesting a mechanism for localizing
p70 S6 kinase to nerve terminals (44).
The 1-subunits SkM1 and SkM2 of voltage-gated sodium channels from
skeletal muscle and heart (45, 46) have recently been shown to bind to
the PDZ domain of
1-syntrophin through their carboxyl-terminal
sequences -KESLV (SkM1) or -RESIV (SkM2) (47, 48). In skeletal muscle
the interaction between SkM1 and
1-syntrophin has been proposed as a
mechanism for anchoring voltage-gated sodium channels in the depths of
the junctional folds of the post-synaptic membrane (46, 47). At the
neuromuscular junction SAPK3 is therefore likely to be anchored in
close proximity to voltage-gated sodium channels.
The carboxyl-terminal sequences of voltage-gated sodium channels
closely resemble the carboxyl-terminal -KETAL or -KETPL of SAPK3 from
different species, except that the terminal leucine is replaced by
valine. However, binding of L367VSAPK3 to the PDZ domain of
1-syntrophin was found to be similar to that of wild-type SAPK3.
Phosphorylation of
1-syntrophin by L367VSAPK3 was also similar to
that of wild-type SAPK3. This indicates that proteins with a leucine
residue at position 0 of the consensus sequence of PDZ domain-binding
proteins will bind to
1-syntrophin. Mammalian type-II activin
receptors are transmembrane serine/threonine protein kinases of the
transforming growth factor
receptor superfamily with the
carboxyl-terminal sequences -KESSL or -KESSI (49, 50), suggesting that
they may also be PDZ domain-binding proteins and bind to
1-syntrophin.
Although SAPK3 is expressed at highest levels in skeletal muscle, it is
expressed at lower levels in many other tissues (20). It is likely that
SAPK3 will be found to interact with the PDZ domains of proteins other
than 1-syntrophin. Possible candidates include the PDZ domains of
proteins whose binding partners have a leucine residue at position 0, such as the recently identified Veli proteins, the vertebrate
homologues of LIN-7 (51). SAPK3 is unique among members of the MAPK
family in having a carboxyl-terminal PDZ domain-binding sequence. It
therefore probably serves distinct physiological functions and is not a
mere isoform of SAPK2a/p38. Inactivation of endogenous SAPK3 by gene
targeting and/or the use of specific inhibitors will help to identify
its specific functions.
Many proteins with PDZ domains localize to specialized cell junctions,
such as synapses and tight junctions, where they bind to the carboxyl
termini of transmembrane proteins, thereby creating a mechanism for
positioning and clustering these proteins and for connecting them to
the cytoplasmic network (52). The finding that SAPK3 co-localizes with
1-syntrophin in skeletal muscle, that it binds to the PDZ domain of
1-syntrophin, and that phosphorylation of
1-syntrophin depends on
this interaction identifies a novel mechanism for targeting a protein
kinase to its substrates. Protein phosphorylation may be important for
modulating the interactions between PDZ domain-containing proteins and
their binding partners. It is also likely that additional protein
kinases that interact with PDZ domains through a carboxyl-terminal
targeting sequence remain to be discovered.
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ACKNOWLEDGEMENTS |
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We thank F. B. Caudwell and N. Morrice
for help with amino acid sequencing and S. C. Froehner for
anti-1-syntrophin antibody SYN17.
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FOOTNOTES |
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* This work was supported by the United Kingdom Medical Research Council (to P. C. and M. G.) and by the Royal Society (to M. G. S. and P. C.).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.
§ Recipient of a post-doctoral fellowship from the Human Frontier Science Program and supported by Innogenetics Inc.
** Awarded a Wellcome Trust Prize studentship.
Supported by a post-doctoral fellowship from Novartis A.G.
§§ To whom correspondence should be addressed. Tel.: 1223-402036; Fax: 1223-213556.
2 M. Hasegawa, A. Cuenda, M. G. Spillantini, G. M. Thomas, V. Buée-Scherrer, P. Cohen, and M. Goedert, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; MBP, myelin basic protein.
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REFERENCES |
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