From the Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received for publication, October 28, 2002
, and in revised form, March 17, 2003.
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ABSTRACT |
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INTRODUCTION |
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Vinexin is a protein localizing at cell-cell and cell-extracellular matrix junctions (9). There are at least two types of vinexin, vinexin and vinexin
, which share a common carboxyl-terminal sequence containing three SH (Src homology) 31 domains. The larger vinexin
has an additional amino-terminal sequence containing a sorbin homology domain. Vinexin is a member of a novel adaptor protein family, including ArgBP2 and ponsin, all of which have a sorbin homology domain in the NH2-terminal half and three SH3 domains in the COOH-terminal half (812). Vinexin binds to vinculin, which also localizes at cell-cell and cell-matrix junctions, through its first and second SH3 domains and enhances actin stress fiber formation and cell spreading (9). Furthermore, vinexin
regulates the anchorage dependence of extracellular signal-regulated kinase activation induced by epidermal growth factor (13, 14). Therefore, vinexin plays a crucial role in regulating cell-extracellular matrix communication, but little is known about the function of vinexin at cell-cell junctions.
Vinculin is a part of the cadherin-catenin junctional complex and is involved in apical junctional organization (1518), suggesting that its binding partner vinexin may have important roles at sites of cell-cell contact. In this study, we identified a membrane-associated guanylate kinase (MAGUK) family protein lp-dlg/KIAA0583 as a vinexin-binding protein. lp-dlg colocalized with vinexin and -catenin at sites of cell-cell contact. lp-dlg was also co-immunoprecipitated with
-catenin. These findings suggest that lp-dlg/KIAA0583 is a scaffolding protein that can link the vinexin-vinculin complex and
-catenin at sites of cell-cell contact.
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EXPERIMENTAL PROCEDURES |
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To define the lp-dlg binding site of vinexin , a deletion mutant containing only the third SH3 domain was excised from plasmids of GST-3rdSH3 (14) and subcloned into pGBT9. Deletion mutants containing the second and third SH3 (amino acids 120328) and the linker (amino acids 173271) region of mouse vinexin
were amplified by polymerase chain reaction and subcloned into pGBT9. pGBT91stSH3, 2ndSH3, and 1st+2ndSH3 were described previously (9). After cotransformation of vinexin deletion mutants in pGBT9 with pMW6 into HF7c, transformants were plated on -Trp, -Leu, -His agar selection media.
PCR-based Full-length lp-dlg cDNA CloningA 5'-rapid amplification of cDNA ends experiment was performed using Human Placenta Marathon Ready (Clontech) cDNA as described previously (19). Primer sets 5'-GACTGATGCCACTGTCTTTCTGTCCAC and AP1 and 5'-CCAGTCATTGACCCTTAAGCGGC and AP2 were used for the first PCR and second PCR, respectively. The several PCR fragments generated by independent PCR were ligated into pCR2.1 (Invitrogen) and sequenced to exclude the fragments containing nucleotide errors introduced by the PCR reaction artificially. The cDNA fragment from one of the longest clones was combined with KIAA0583 cDNA (provided by Dr. Takahiro Nagase) using AflII to construct the full-length lp-dlg cDNA. The resulting full-length cDNA was subcloned into pGZ21 (9) for expressing as GFP-tagged protein.
AntibodiesThe cDNA insert of the clone (pMW6) isolated by the yeast two-hybrid screening was subcloned into pGEX4T-1 (Amersham Biosciences) for expressing as GST-tagged protein and designed pGST-MW6. Rabbit anti-lp-dlg antiserum was raised against GST-MW6. Polyclonal antibodies were affinity purified using GST-MW6 covalently conjugated to Affi-Gel 10 (Bio-Rad) followed by the adsorption with Affi-Gel 10 conjugated with GST. Anti-FLAG antibody M2, anti-GFP antibody, and anti--catenin antibody were obtained from Sigma, Santa Cruz Biotechnology, and Transduction Laboratories, respectively. Anti-HA antibodies were purchased from Roche Diagnostics and Santa Cruz Biotechnology.
Northern BlottingA cDNA fragment of 11557 base pairs of the lp-dlg coding region was radiolabeled using a Random Primer DNA labeling kit version 2.0 (TAKARA) and then used to probe the Human Multiple Northern blot (Clontech) containing 2 µg of poly(A)+ RNA, as described previously (9).
In Vitro Binding Assay Using Affinity PrecipitationThe GST-fused proteins containing vinexin deletion mutants, GST-1stSH3, GST-2ndSH3, GST-3rdSH3, GST-3rdSH3WF, and GST-3rdSH3YV were described previously (14). Full-length vinexin was subcloned into pGEX4T-1. The cDNA insert of pMW6 was subcloned into p401F (9) to make FLAG-tagged MW6. To construct FLAG-tagged lp-dlg deletion mutants (644909, 842909, 842879, and 880909), the corresponding region of lp-dlg was amplified by PCR and subcloned into p401F. In vitro binding assays were performed as described previously (12). In brief, COS-7 cells were transiently transfected with various FLAG-tagged constructs and washed twice with phosphate-buffered saline and lysed in Triton lysis buffer (1% Triton X-100, 100 µg/ml p-amidinophenylmethanesulfonyl fluoride hydrochloride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). The lysates were incubated with 3 µg of each GST fusion protein and glutathione-Sepharose 4B (Amersham Biosciences) at 4 °C for 3 h. After four washes with lysis buffer, co-precipitated proteins were resolved by 8% SDS-PAGE and analyzed by Western blot with anti-FLAG M2 antibody.
ImmunoprecipitationCOS-7 cells were transiently transfected with p401F-MW6 with or without expression plasmid for GFP-tagged vinexin (9). COS-7 cells were lysed as described above and equal amounts of total proteins were incubated with 5 µg of anti-FLAG antibody M2 for 1 h at 4 °C. The immunocomplexes were incubated with protein G-Sepharose for 1 h and washed four times with lysis buffer. The bound proteins were detected as described above with anti-GFP antibody. To examine the interaction of FLAG-vinexins with endogenous lp-dlg, FLAG-tagged vinexin genes were subcloned into the pLRT-X, which was designed for the tetracycline-On (Tet-ON) expression system (20). Then the plasmids were stably transfected into LLC-PK1 cells. After inducing the expression of FLAG-tagged vinexin
and
by adding the tetracycline derivative doxycycline (1.5 µg/ml), immunoprecipitation was performed as described above, followed by immunoblotting using anti-lp-dlg antibody.
To detect the interaction of lp-dlg and -catenin, FLAG-tagged lp-dlg mutants were transfected into COS-7 cells with GFP-tagged
-catenin. Cells were then lysed and immunoprecipitated with anti-FLAG M2 antibody as described above. The bound proteins were detected using anti-GFP antibody. To detect the interaction of endogenous lp-dlg with
-catenin, endogenous lp-dlg from LLC-PK1 cells was immunoprecipitated with anti-lp-dlg antibody. Co-precipitated
-catenin was detected as described above using anti-
-catenin antibody.
ImmunostainingFor immunostaining of endogenous lp-dlg with GFP-vinexin , LLC-PK1 cells were transfected with GFP-tagged vinexin
using LipofectAMINE (Invitorgen). The cells were fixed with methanol at room temperature for 1 min. For co-immunostaining of endogenous lp-dlg and
-catenin, LLC-PK1 cells were fixed with acetone at room temperature for 30 s. Immunofluorescence staining was carried out as described previously (9). The fluorescence images were obtained using an Axiovert microscope (Carl Zeiss) equipped with a MicroRadiance confocal laser scanning microscope (Bio-Rad).
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RESULTS |
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To confirm the expression of lp-dlg in cultured cells, Western blot analysis was performed using anti-lp-dlg polyclonal antibody. Two major protein bands of 250 and 200 kDa were detected at high levels in LLC-PK1 cells and moderately in other cell lines (Fig. 2B). Both proteins were also detected by another anti-lp-dlg antibody purified from a different rabbit serum (data not shown), suggesting that they are isoforms. To determine which proteins are translated from lp-dlg mRNA, full-length cDNA of lp-dlg was transfected into COS-7 cells. Proteins of 250 kDa were detected in addition to endogenous proteins (data not shown), suggesting that the 250-kDa protein is translated from lp-dlg mRNA and that the 200-kDa protein is from another splicing variant of lp-dlg.
Interaction of Vinexin with lp-dlgTo determine the region of the lp-dlg binding site in vinexin, various deletion mutants of vinexin were fused with the GAL4 DNA-binding domain (Fig. 3A). The two-hybrid system using these deletion mutants as bait plasmids and pMW6 as a prey plasmid were performed. Transformants containing vinexin , 2nd+3rdSH3, and 3rdSH3 showed histidine autotrophy, suggesting that vinexin
binds to lp-dlg through its third SH3 domain (Fig. 3B). To further confirm the interaction of vinexin
with lp-dlg in vitro, various SH3 domains of vinexin
were expressed and purified as GST fusion proteins. These GST fusion proteins were immobilized on a glutathione-Sepharose 4B and incubated with cell lysates from COS-7 cells expressing FLAG-MW6. The bound proteins were analyzed by immunoblotting using an anti-FLAG antibody. Consistent with the results of the yeast two-hybrid system, GST-vinexin
and GST-3rdSH3 were able to interact with FLAG-MW6 (Fig. 4A). In contrast, neither GST-1stSH3 nor GST-2ndSH3 bound to FLAG-MW6 (Fig. 4B), suggesting that the interaction of the third SH3 domain of vinexin and lp-dlg is specific. Furthermore, the 3rdWF mutant (tryptophan residue at position 306 to phenylalanine) and the 3rdYV mutant (tyrosine residue at position 324 to valine), which lost the binding ability to the target protein Sos (14), could not interact with FLAG-MW6 (Fig. 4B). Together these results suggest that lp-dlg binds to vinexin specifically and that the third SH3 domain of vinexin was responsible for this interaction.
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To map the vinexin binding site in lp-dlg, various deletion mutants of lp-dlg (Fig. 5A) tagged with the FLAG epitope were transfected into COS-7 cells. These mutants were expressed comparably in COS-7 cells (Fig. 5B). Cell lysates were then incubated with GST-vinexin . Immunoblotting using anti-FLAG antibody against the bound proteins showed that vinexin
interacted with FLAG-MW6, 644909, 842909, and 842879 strongly but not 880909 (Fig. 5B). Similar results were also obtained using GST-3rdSH3 in place of GST-vinexin
(data not shown). These results suggest that the third SH3 domain of vinexin
binds to the region of 842879 of lp-dlg, which contains proline-rich putative SH3 binding sequences.
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To confirm the in vivo association of vinexin with lp-dlg, co-immunoprecipitation experiments were performed. GFP-tagged vinexin
was transfected with or without FLAG-MW6 into COS-7 cells. Equal amounts of total protein lysates were immunoprecipitated using anti-FLAG antibody. As shown in Fig. 6A, GFP-vinexin
was co-precipitated with FLAG-MW6. To verify the interaction of vinexin with full-length lp-dlg, FLAG-tagged vinexin
and
were expressed in LLC-PK1 cells. Total cell lysates were immunoprecipitated using anti-FLAG antibody, and co-precipitated endogenous lp-dlg was examined by immunoblotting using anti-lp-dlg antibody. Precipitated lp-dlg was detected in cells expressing both FLAG-tagged vinexin
and
, but not in LLC-PK1 (Fig. 6B). Together these observations suggest that vinexin
interacts with lp-dlg both in vitro and in vivo.
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Subcellular Localization of lp-dlgTo determine whether lp-dlg and vinexin are colocalized at sites of cell-cell contact, GFP-tagged vinexin was transfected into LLC-PK1 cells where lp-dlg was expressed at a high level. The subcellular localization of vinexin
was observed by GFP fluorescence, and the same cells were also stained with anti-lp-dlg polyclonal antibody and Alexa 568-labeled secondary antibody. GFP-tagged vinexin
showed the localization at both sites of cell-cell and cell-extracellular matrix junctions, in addition to a diffuse pattern (Fig. 7A). Endogenous lp-dlg was also concentrated at sites of cell-cell contact and colocalized with vinexin
(Fig. 7A). Interestingly, lp-dlg was not concentrated at sites of cell-extracellular matrix junction (Fig. 7A, inset). Furthermore, lp-dlg was partially colocalized with
-catenin, a major component of adherens junctions (Fig. 7B). We also examined the localization of lp-dlg protein in frozen tissue sections of mouse placenta, where lp-dlg mRNA was expressed at high levels (Fig. 2A), and found that lp-dlg and
-catenin were also colocalized in tissues (data not shown).
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Interaction of lp-dlg with -CateninCo-localization of lp-dlg and
-catenin both in cultured cells and in tissues raises the possibility of the interaction between these two molecules. To examine this possibility, FLAG-tagged lp-dlg mutants were transiently expressed in COS-7 cells by transfection with GFP-tagged
-catenin. Equal amounts of total protein lysates were immunoprecipitated using anti-FLAG antibody. As shown in Fig. 8A, GFP-
-catenin was co-precipitated with FLAG-MW6 but not with vector alone, suggesting that lp-dlg can associate with
-catenin. FLAG-MW6 also contained the binding site (842879) for vinexin
as described above (see Fig. 5B). To determine whether vinexin
and
-catenin share the binding site in lp-dlg, a co-immunoprecipitation experiment using another deletion mutant (FLAG-(644909)) was performed (Fig. 8A). FLAG-(644909) contains the binding site for vinexin
but not the first and second PDZ domains. GFP-
-catenin was not co-immunoprecipitated with FLAG-(644909). These results suggest that the binding site of lp-dlg for
-catenin is different from that for vinexin
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To confirm the interaction of lp-dlg with -catenin, endogenous lp-dlg was immunoprecipitated using anti-lp-dlg polyclonal antibody from lysates of LLC-PK1 cells, in which lp-dlg was expressed at high levels. Precipitates were evaluated by immunoblotting with anti-
-catenin antibody. Fig. 8B showed that endogenous
-catenin was co-immunoprecipitated with endogenous lp-dlg.
-Catenin was not detected in the precipitate using preimmune serum. E-cadherin and
-catenin showed very weak association with lp-dlg compared with that of
-catenin (data not shown). These results suggest that lp-dlg associates with
-catenin as well as vinexin at sites of cell-cell contact.
To determine whether lp-dlg forms a ternary complex with vinexin and
-catenin, GFP-vinexin
and HA-
-catenin were transiently expressed in COS-7 cells with or without FLAG-MW6, and then immunoprecipitated with anti-HA antibody. GFP-vinexin
was barely co-precipitated with HA-
-catenin without FLAG-MW6 (Fig. 8C). However, GFP-vinexin
was co-precipitated significantly with HA-
-catenin when FLAG-MW6, which includes the binding sites for both vinexin
and
-catenin, was coexpressed (Fig. 8C). This observation suggests that lp-dlg can form a ternary complex with vinexin
and
-catenin and function as a scaffolding protein.
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DISCUSSION |
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-Catenin connects the cytoplasmic domain of cadherin to
-catenin, which then binds to the actin cytoskeleton directly or indirectly thorough vinculin. In this study, we showed that both the endogenous and the deletion mutants of lp-dlg can bind to
-catenin. The region (472644) containing the first and second PDZ domains of lp-dlg were required for this association.
-Catenin has been shown to have a PDZ-target like sequence (-DTDL) at its COOH-terminal end and to bind to proteins containing PDZ domains (24, 25). Thus, the first or second PDZ domain of lp-dlg may mediate the interaction of lp-dlg with
-catenin. We also showed that lp-dlg can form a ternary complex with
-catenin and vinexin
. The function of this complex is unclear so far. However, it is possible that the
-catenin·lp-dlg·vinexin complex can link cadherin to actin cytoskeleton through vinexin binding to vinculin and contribute to the formation of cell-cell contacts. Alternatively,
-catenin·lp-dlg complex might compete with the vinexin binding to Sos, a guanine nucleotide exchange factor for Ras and Rac, and modulate the signaling, because both Sos and lp-dlg bind to the third SH3 domain of vinexin
(14). Further studies are necessary to examine these possibilities.
lp-dlg contains an NH2-terminal coiled-coil region, four PDZ domains, an SH3 domain, and a GUK domain, and belongs to the MAGUK protein family. This domain structure is slightly different from typical MAGUK proteins, which have one or three PDZ domains and no coiled-coil regions. In addition, none of the four PDZ domains of lp-dlg have the GLGF motif, which is conserved in most PDZ domains and is necessary for binding to S/T-X- (
is a hydrophobic residue). A recently proposed classification (26) also classified the four PDZ domains of lp-dlg into different groups from the PDZ domains of typical MAGUK proteins. Thus, lp-dlg may have different target proteins and different functions from other MAGUK proteins.
We showed that the third SH3 domain of vinexin bound to the region of 842879 of lp-dlg. This binding was specific, because neither the first nor the second SH3 domain of vinexin
bound to lp-dlg. In addition, the result that point mutations in the third SH3 domains disrupted the binding ability to lp-dlg suggests the specific interaction, although other proteins containing SH3 domain may also bind to lp-dlg. The region of 842879 of lp-dlg included the proline-rich sequences, RagPlt-PPkPPRR. Two sequence motifs, RXXPXXP and PXXPXR, were reported to be a consensus for binding to SH3 domains (27). Interestingly, the proline-rich sequences in 842879 of lp-dlg contain both consensus motifs, suggesting that they mediate the interaction with the third SH3 domain of vinexin
.
During preparation of the present manuscript, another splicing variant of lp-dlg, DLG5, was reported (28). DLG5 was isolated from the heart as one of the genes located on chromosome 10q22, where familial atrial fibrillation was mapped, and excluded as a possible cause of familial atrial fibrillation (28). DLG5 contains an additional 45 amino acids at the NH2-terminal compared with lp-dlg. Both DLG5 and lp-dlg cDNA contain stop codons upstream of their first ATG in-frame, suggesting that both products include whole coding regions of each product. lp-dlg was isolated from placenta, where lp-dlg/DLG5 expression is high, and DLG5 from the heart, where lp-dlg/DLG5 expression is lower (Fig. 2). Thus, lp-dlg and DLG5 may represent the splicing variant expressed in the placenta and heart, respectively.
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FOOTNOTES |
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To whom correspondence should be addressed. Fax: 81-75-753-6104; E-mail: nkioka{at}kais.kyoto-u.ac.jp.
1 The abbreviations used are: SH3, Src homology 3; ERK, extracellular signal-regulated kinase; MAGUK, membrane-associated guanylate kinase; GST, glutathione S-transferase; HA, hemagglutinin; GFP, green fluorescent protein.
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ACKNOWLEDGMENTS |
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REFERENCES |
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