Stress-activated Protein Kinase-3 Interacts with the PDZ Domain of alpha 1-Syntrophin
A MECHANISM FOR SPECIFIC SUBSTRATE RECOGNITION*

Masato HasegawaDagger §, Ana Cuenda, Maria Grazia Spillantiniparallel , Gareth M. Thomas**, Valérie Buée-ScherrerDagger Dagger Dagger , Philip Cohen, and Michel GoedertDagger §§

From the Dagger  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 parallel  Department of Neurology, E.D. Adrian Building, University of Cambridge, Robinson Way, Cambridge CB2 2PY, United Kingdom

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 p38gamma ), 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 alpha 1-syntrophin. SAPK3 phosphorylates alpha 1-syntrophin at serine residues 193 and 201 in vitro and phosphorylation is dependent on binding to the PDZ domain of alpha 1-syntrophin. In skeletal muscle SAPK3 and alpha 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

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 p38beta 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 (p38beta 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).

A third class of SAPK consists of the more recently identified SAPK3 (also called ERK6 and p38gamma ) (20-23) and SAPK4 (also called p38delta ) (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 alpha 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 alpha 1-syntrophin. In skeletal muscle SAPK3 and alpha 1-syntrophin were found to co-localize at the neuromuscular junction and throughout the sarcolemma.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Full-length human alpha 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. alpha 1-Syntrophin (78-179) and alpha 1-syntrophin (174-505) were produced by polymerase chain reaction, as were human beta 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).

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-, Trp-, His-) + 25 mM 3-aminotriazole and assayed for beta -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.

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-alpha 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 [gamma -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 alpha 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 alpha 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.

Immunofluorescence-- Pectoral and semitendinous muscles were dissected from five adult Sprague-Dawley rats and kept at -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 alpha -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-alpha 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 alpha -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 alpha -bungarotoxin and antibodies R5 or SYN17 were alternatively omitted.

Transfection and Immunoprecipitation-- Full-length rat SAPK3 and human alpha 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 alpha 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 beta -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-alpha 1-syntrophin serum TROPHA. TROPHA was raised in a rabbit against the synthetic peptide ASGRRAPRTGLLELRAG (corresponding to residues 2-17 of human alpha 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-alpha 1-syntrophin serum TROPHA and anti-SAPK3 serum R5.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 1-syntrophin. alpha 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 beta 1-syntrophin and beta 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 alpha 1-syntrophin-SAPK3 interaction (Fig. 1). Full-length alpha 1-syntrophin interacted with SAPK3. The shortest construct that was positive when paired with SAPK3 contained the PDZ domain (residues 78-179) of alpha 1-syntrophin. By contrast, a construct extending from the end of the PDZ domain to the carboxyl terminus of alpha 1-syntrophin (residues 174-505) failed to interact with SAPK3, establishing that the PDZ domain of alpha 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 alpha 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 alpha 1-syntrophin. Similarly, SAPK3 interacted with the PDZ domain of beta 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 alpha 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 alpha 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 alpha 1-syntrophin. A, interaction of SAPK3 with the PDZ domain of alpha 1-syntrophin. Binding of GAL4 fusion constructs of human alpha 1-syntrophin, the PDZ domain of human beta 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 beta -galactosidase. HIS3 activity was judged by growth in medium lacking histidine in the presence of 25 mM 3-aminotriazole and beta -galactosidase activity was determined from the time taken for the colonies to turn blue in 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside filter lift assays performed at room temperature: +, 90-240 min; -, no significant beta -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 alpha 1-syntrophin. B, interaction of human alpha 1-syntrophin with full-length rat SAPK3(1-367) but not with SAPK3(1-363).

Human alpha 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-alpha 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 alpha 1-syntrophin is a good substrate for SAPK3 but not for other SAPKs or for p42 MAPK (Table I). SAPK3 phosphorylated alpha 1-syntrophin at approximately the same rate as it phosphorylated MBP, its standard substrate (Table I). Phosphorylation of alpha 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). alpha 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 alpha 1-syntrophin but not MBP (Fig. 2C). Finally, preincubation of alpha 1-syntrophin with synthetic peptides corresponding to the carboxyl-terminal 6 or 8 amino acids of rat SAPK3 prevented phosphorylation of alpha 1-syntrophin by GST-SAPK3 (Fig. 2D).


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Fig. 2.   Phosphorylation of alpha 1-syntrophin by SAPK3 is dependent on the carboxyl-terminal four amino acids of SAPK3. A, GST-alpha 1-syntrophin phosphorylated by SAPK3 was digested with trypsin, and the resulting phosphopeptides were chromatographed on a C18 column (see "Experimental Procedures"). The two major tryptic phosphopeptides were shown to correspond to residues 198-207 and 178-197 and to be phosphorylated at Ser193 and Ser201, respectively. The acetonitrile gradient is shown by the broken line. B, GST-alpha 1-syntrophin (1 µM) or MBP (1 µM) was phosphorylated for the times indicated with 0.2 units/ml of either GST-SAPK3(1-367) or GST-SAPK3(1-363). The results are shown as the means ± S.E. from three experiments. C, full-length GST-SAPK3 (0.2 units/ml) was incubated for 30 min at room temperature with the indicated concentrations of an antibody raised in sheep against the synthetic peptide KPPRQLGARVPKETAL, which corresponds to residues 352-367 of rat SAPK3 (open symbols) (20) or with sheep IgG (closed symbols). The SAPK3 was then assayed in duplicate for 10 min with GST-alpha 1-syntrophin (circles) or MBP (triangles). Substrate phosphorylation is plotted as a percentage of that measured in the absence of antibody. D, GST-alpha 1-syntrophin (filled bars) or MBP (open bars), each at 1 µM, were incubated for 30 min at room temperature with synthetic peptides (300 µM) corresponding to the carboxyl-terminal 6 or 8 amino acids of rat SAPK3. GST-SAPK3 was added to 0.2 units/ml, and after 10 min the assays were initiated with Mg[gamma -32P]ATP. Substrate phosphorylation is plotted as a percentage of that measured in the absence of each peptide. The concentration of each peptide required to inhibit GST-alpha 1-syntrophin phosphorylation by 50% was 30 µM. The results are shown as the means ± S.E. from a single experiment. The assays in C and D were carried out in duplicate, and similar results were obtained in two further experiments.

                              
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Table I
Comparison of substrate specificities of different MAPK family members (assayed as in Fig. 2)

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-alpha 1-syntrophin. By ELISA, the binding of wild-type GST-SAPK3 to alpha 1-syntrophin was similar to that of mutant GST-L367VSAPK3 (Fig. 3A). The rate of phosphorylation of alpha 1-syntrophin by GST-L367VSAPK3 was slightly faster than by wild-type GST-SAPK3 (Fig. 3B). However, both mutant and wild-type SAPK3 phosphorylated alpha 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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Fig. 3.   Binding of L367V SAPK3 to alpha 1-syntrophin and phosphorylation of alpha 1-syntrophin by L367VSAPK3. A, in vitro binding of wild-type SAPK3 and L367VSAPK3 to alpha 1-syntrophin as determined by ELISA. B and C, GST-alpha 1-syntrophin (1 µM) (B) or MBP (1 µM) (C) was phosphorylated for the times indicated with 0.2 units/ml of either wild-type GST-SAPK3 or GST-V367LSAPK3.

If the association of SAPK3 with alpha 1-syntrophin is physiologically relevant, the two proteins should be co-localized in vivo. Both SAPK3 and alpha 1-syntrophin are expressed at highest levels in skeletal muscle (20-22, 30, 31), where alpha 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 alpha -bungarotoxin, which visualizes nicotinic acetylcholine receptors at the neuromuscular junction (Fig. 4). Moreover, double staining for SAPK3 and alpha 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 alpha 1-syntrophin-SAPK3 interaction, the ability of SAPK3 to co-immunoprecipitate with alpha 1-syntrophin was examined in extracts from mammalian cells co-transfected with both proteins. alpha 1-Syntrophin and SAPK3 were co-expressed transiently in COS cells. Immunoprecipitation was carried out using an anti-alpha 1-syntrophin antibody, and proteins present in the pellet were immunoblotted using anti-alpha 1-syntrophin and anti-SAPK3 antibodies. The strong signal seen for SAPK3 upon immunoprecipitation with the anti-alpha 1-syntrophin antibody indicates that alpha 1-syntrophin existed in a complex with SAPK3 in COS cell lysates (Fig. 5).


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Fig. 4.   Localization of SAPK3, alpha -bungarotoxin, and alpha 1-syntrophin in rat skeletal muscle. Sections of semitendinous muscle were double or triple stained with tetramethylrhodamine alpha -bungarotoxin (in red) (A), anti-SAPK3 serum R5 visualized with fluorescein-avidin D (in green) (B) and anti-alpha 1-syntrophin serum SYN17 visualized with 7-amino-4-methylcoumarin-3-acetic acid-streptavidin (in blue) (C). alpha -Bungarotoxin, SAPK3, and alpha 1-syntrophin are present at the neuromuscular junction. SAPK3 and alpha 1-syntrophin are also present throughout the sarcolemma (B and C). Scale bar, 17 µm.


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Fig. 5.   Co-immunoprecipitation of SAPK3 with alpha 1-syntrophin. Lysates from COS cells transfected with rat SAPK3 alone (marked SAPK3) or double transfected with SAPK3 and human alpha 1-syntrophin (marked SAPK3 + alpha 1-Syntrophin) were immunoprecipitated with anti-alpha 1-syntrophin serum TROPHA. Total cell lysates and immunoprecipitates (marked IP) were immunoblotted with anti-alpha 1-syntrophin and anti-SAPK3 antibodies. alpha 1-Syntrophin-immunoreactive bands were present in double transfected cell lysates and immunoprecipitates (arrows). SAPK3-immunoreactive bands were detected in single and double transfected cell lysates (arrowhead). SAPK3 was detected as an immune complex with alpha 1-syntrophin in double transfected cell lysates (arrowhead) but not in cells transfected with SAPK3 alone. The strong band in the lanes marked IP corresponds to the IgG heavy chain.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

SAPK3 is a protein kinase whose phosphorylation of alpha 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 alpha  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 alpha -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 alpha 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 alpha 1-syntrophin through their carboxyl-terminal sequences -KESLV (SkM1) or -RESIV (SkM2) (47, 48). In skeletal muscle the interaction between SkM1 and alpha 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 alpha 1-syntrophin was found to be similar to that of wild-type SAPK3. Phosphorylation of alpha 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 alpha 1-syntrophin. Mammalian type-II activin receptors are transmembrane serine/threonine protein kinases of the transforming growth factor beta  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 alpha 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 alpha 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 alpha 1-syntrophin in skeletal muscle, that it binds to the PDZ domain of alpha 1-syntrophin, and that phosphorylation of alpha 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.

    ACKNOWLEDGEMENTS

We thank F. B. Caudwell and N. Morrice for help with amino acid sequencing and S. C. Froehner for anti-alpha 1-syntrophin antibody SYN17.

    FOOTNOTES

* 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.

Dagger Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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