The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
* Present address: Department of Dermatology, Geneva University Hospital, 24 rue Micheli-Ducrest, 1211 Geneva 4, Switzerland
Present address: Department of Human Genetics M1-159, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
L. Fontao and D. Geerts contributed equally to this work
¶Author for correspondence (e-mail: asonn{at}nki.nl)
Accepted March 9, 2001
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SUMMARY |
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Key words: Plectin, Dystrophin, Actin-binding domain, Focal contact
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INTRODUCTION |
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Sequence analysis of human (McLean et al., 1996) and rat (Wiche et al., 1991) plectin cDNA revealed extensive homology between plectin and other intermediate filament associated proteins (IFAPs), including desmoplakin and the bullous pemphigoid antigen BP230. Based on these sequence homologies, plectin, desmoplakin and BP230 were classified as members of a new family of proteins involved in connecting IFs to the plasma membrane, the plakins (Ruhrberg and Watt, 1997). Each of these proteins consists of a long central -helical, coiled-coil rod domain flanked by globular end domains. The plectin C-terminal globular domain contains repeated sequences organized in six subdomains (R1-R6). Transfection studies with cDNAs encoding truncated plectin molecules have revealed that the intermediate filament-binding domain (IFBD) of plectin resides in a stretch of 50 amino acids within the R5 repeat that serves as the unique binding site for vimentin and keratin filaments (Nikolic et al., 1996). Extending these findings we have recently shown, by a yeast two-hybrid assay, that a Gal4-fusion protein containing the IFBD of plectin can efficiently bind GFAP, vimentin and monomeric keratins 14 and 18, but not keratins 5 and 8 (Geerts et al., 1999).
The precise function of the central rod domain of plectin has not yet been established, but there is evidence that this domain mediates plectin dimerization and/or multimerization (Foisner and Wiche, 1987; Uitto et al., 1996). By RT-PCR, mRNA encoding a rod-less plectin was identified in several different rat tissues (Elliott et al., 1997). However, whether this particular mRNA is translated in vivo into a functional variant of plectin is not known. Although it was known for a long time that plectin can be colocalized with actin stress fibers in focal adhesions (Seifert et al., 1992; Sánchez-Aparicio et al., 1997), evidence for a direct binding between plectin and actin has only recently been obtained. Sequence analysis reveals an actin-binding domain (ABD) of the ß-spectrin type in the N-terminal globular domain of plectin (McLean et al., 1996). This ABD is also found in other actin-binding proteins, such as dystonin, fimbrin, -actinin and dystrophin (for a review, see Hartwig, 1994), and is composed of two consecutive calponin homology (CH) domains. Biochemical studies have identified three potential actin-binding sequences (ABS) within this ABD, termed ABS1, ABS2 and ABS3. The ABS1 and ABS3 were first identified by NMR studies of dystrophin, which showed that synthetic peptides corresponding to these two sequences bind to actin. ABS2 was originally identified in ABP120 (a Dictyostelium actin-gelation factor) in which it is required to mediate binding to actin (Bresnick et al., 1990).
In plectin-deficient mouse fibroblasts, an N-terminal fragment of plectin that contains the ABD was found to decorate actin stress fibers (Andrä et al., 1998). We have shown that the N-terminal fragment of plectin also interacts with the cytoplasmic domain of the ß4 integrin subunit and that binding of ß4 to plectin prevents actin from interacting with it (Geerts et al., 1999). Furthermore, plectin regulates actin dynamics and is involved in the reorganization of the actin cytoskeleton in response to activation of small GTPases (Andrä et al., 1998). A role of plectin in actin dynamics is also suggested by the finding that in plectin-deficient fibroblasts, as compared to wild-type cells, the reorganization of the actin cytoskeleton induced by CD95-mediated apoptosis was severely impaired (Stegh et al., 2000). The molecular mechanism by which plectin regulates actin dynamics remains unclear.
The aim of this study was to investigate which domains are involved in the specific interaction of plectin with the ß-cytoplasmic actin isoform. To further establish the multifunctional role of plectin as a cytoskeletal linker protein, we have investigated whether plectin can also bind the non-muscular -cytoplasmic actin and the
-skeletal muscle actin. Whether plectin-ABD can bind G- and/or F-actin was studied using an in vitro binding assay and the effects of plectin-ABD on actin polymerization were investigated by kinetic analysis and electron microscopy. Our results show that plectin can dimerize by its ABD, and that this leads to the bundling of actin filaments. Furthermore, we identified dystrophin and dystonin as additional actin-binding proteins that can homodimerize by their ABD and show that plectin and dystonin can also form heterodimers by their ABD.
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MATERIALS AND METHODS |
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For use in cell transfection experiments, cDNA fragments encoding plectin1-339 and plectin65-339 were isolated from pAS2-1 constructs (see above) and inserted into pcDNA3HA (Geerts et al., 1999). Similarly, a cDNA fragment encoding plectin1-339 was inserted into the bacterial maltose binding protein (MBP)-fusion protein expression vector pMAL-c2X (New England Biolabs Inc.), for the production of MBP-fusion proteins, or in pRP261, a derivative of pGEX-3x (Amrad Corp. Ltd), for the production of glutathione S-transferase (GST)-fusion proteins. The ß4 integrin cDNA expression construct used for the experiments in Fig. 5 encoded ß4A integrin cytoplasmic domain from residues 1115-1449 with a single amino acid substitution R1281W (ß4R1281W), which abrogates plectin binding, as previously described (Geerts et al., 1999).
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Purification of recombinant fusion proteins
The E. coli strain BL21(DE3) (Novagen) was transformed with recombinant plasmids and colonies obtained were used to inoculate Luria Bertani medium containing 100 µg/ml ampicillin; cultures were grown as previously described (Geerts et al., 1999). Bacteria were harvested by centrifugation at 4,000 g, resuspended in PBS containing 1 mM EDTA and 1% (v/v) Triton X-100, and lysed by sonification. Lysates were cleared by centrifugation for 10 minutes at 10,000 g and 4°C, and the resulting supernatants were incubated with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). Beads with affinity-bound proteins were washed three times with PBS containing 1% (v/v) Triton X-100, and equilibrated in 50 mM Tris-HCl (pH 8.0). Bound proteins were eluted in 50 mM Tris-HCl (pH 8.0) containing 10 mM reduced glutathione. Recombinant MBP-plectin-ABD fusion-protein was expressed and purified as described above, except that amylose resin (800-21, New England Biolabs) was used for the affinity purification, and that equilibration and elution of the resin was in 20 mM Tris-HCl (pH 7.4), 1 mM ß-mercaptoethanol without or with 10 mM maltose, respectively.
Buffers containing the eluted fusion-proteins were exchanged in actin-G buffer (AGB: 2 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.5 mM ATP) by dialysis and protein concentration was estimated by the Bradford protein assay (Biorad).
Actin-binding assay
All purified proteins used in this study were clarified by centrifugation at 100,000 g for 1 hour at 4°C and kept on ice in AGB. Actin cosedimentation assays were performed as follows: rabbit -skeletal muscle actin (2.5 µM; Cytoskeleton Inc.), premixed or not premixed with fusion proteins, was allowed to polymerize by the addition of 0.1 volume of 10x initiation mix (IM: 2 mM Tris-HCl, pH 8.0, 20 mM MgCl2, 1 M KCl, 5 mM ATP) for 1 hour at room temperature. Actin filaments with bound proteins were pelleted by centrifugation at 100,000 g for 1 hour at 20°C. Equal amounts of pellet and supernatant were resolved by SDS-PAGE and proteins were visualized by Coomassie Brilliant Blue-staining.
To test the ability of plectin-ABD to bind monomeric actin, MBP-plectin1-339 bound to amylose resin was equilibrated in AGB. Monomeric actin was mixed with immobilized MBP-plectin1-339 to obtain a final concentration of 2.5 µM for actin and 3 µM for plectin. The mixture was incubated for 3 hours at room temperature. After five washes with AGB, samples were boiled in sample buffer and appropriate amounts of bound and unbound protein mixture were resolved by SDS-PAGE. Proteins were visualized by Coomassie Brilliant Blue-staining.
Low-speed sedimentation assays were performed as follows. Actin was polymerized at 24 µM for 3 hours at room temperature, then diluted to 4.5 µM in actin polymerization buffer (APB: AGB supplemented with 0.1 volume of IM) containing 2, 1 or 0.5 µM of MBP-plectin or MBP alone (control). After a 1 hour incubation at room temperature, samples were centrifuged at 14,000 g for 1 minute, equal amounts of pellet and supernatant were analyzed by SDS-PAGE and proteins visualized by Coomassie Brilliant Blue-staining.
Actin filament assembly assays and electron microscopy
For the pyrene-actin assembly assays (Kouyama and Mihashi, 1981), an actin polymerization kit was purchased from Cytoskeleton Inc. Rabbit -skeletal muscle actin containing 10% (mol/mol) of pyrene-labeled actin was used at a final concentration of 4 µM. Polymerization was induced by the addition of 0.1 volume of IM and monitored by change in fluorescence at an excitation wavelength of 365 nm and emission wavelength of 407 nm in a fluorimeter. The fluorescence corresponding to 100% of polymerization was measured 3-4 hours after the addition of the IM. Light scattering assays were performed with unlabeled actin (2 µM) by monitoring changes in OD at 300 nm upon the addition of the IM. Electron microscopy was performed on samples after the actin polymerization was complete, as monitored by light scattering. Samples of the polymerization mixtures were fixed by the addition of 0.05% (v/v) glutaraldehyde, spotted on formvar-carbon coated grids, negatively stained with 1% (w/v) aqueous uranyl acetate and examined at 80 kV with a Philips CM 10 electron microscope.
Cell culture, transfection and immunofluorescence microscopy
Rat embryo fibroblasts (REF) and COS-7 cells were maintained in DMEM (Gibco BRL) containing 10% foetal calf serum (FCS) and supplemented with 100 i.u./ml penicillin and 100 U/ml streptomycin. Cells were grown at 37°C in a humidified, 5% CO2 atmosphere. REFs were transiently transfected with cDNA constructs using Lipofectin (Gibco BRL) according to the manufacturers instructions. COS-7 cells were transfected using DEAE-dextran as previously described (Schaapveld et al., 1998). Indirect immunofluorescence staining of transfected REF cells with mouse mAb 12CA5 against the haemaglutinin (HA)-epitope (YPYDVPDYA) (Santa Cruz Biotechnology) was performed as described previously (Geerts et al., 1999). Actin filaments were stained with rhodamine-phalloidin from Molecular Probes. Immunfluorescence images were taken using a Leica confocal laser scanning microscope.
In vitro binding assays
The ability of the plectin-ABD to interact with another plectin-ABD was tested in an in vitro binding assay with radiolabeled proteins. Coupled in vitro transcription-translation of 1-2 µg of pcDNA3HA-plectin-ABD1-339 or pcDNA3HA-ß4R1281W (negative control) was performed using the TnT rabbit reticulocyte lysate kit (Promega) in the presence of [35S]-methionine/cysteine. Non-incorporated radiolabeled amino acids were removed from the in vitro translation mixture by gel filtration using a PD10 column (Amersham Inc.). Scintillation counting performed on the purified translation mixtures indicated that both proteins were equally labeled, with 9% of the total [35S]-methionine/cysteine incorporated. Purified translation mixtures were then tested for binding to MBP-plectin1-339, or MBP (negative control) immobilized on amylose-agarose beads. For the binding assay, the translation mixtures were diluted in APB containing 2% of heat-inactivated BSA (HI-BSA: BSA heated overnight at 55°C) and incubated for 2 hours at room temperature with 200 pmol of MBP-plectin or MBP immobilized on amylose beads. After incubation, beads were washed 3 times in APB supplemented with 2% (w/v) HI-BSA, then twice in APB. After washing, the beads were boiled in sample buffer, proteins were subjected to SDS-PAGE and visualized by autoradiography.
A pull-down assay was used to study plectin-ABD interaction in mammalian cells. 48 hours after transfection, COS-7 cells were lysed in ice-cold lysis buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1% (v/v) Nonidet P-40, 1 mM MgCl2, 1 mM CaCl2) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and soybean trypsin inhibitor and 0.1 U/ml aprotinin). Lysates were clarified by centrifugation at 4°C (20 minutes at 15,000 g) and diluted five times in buffer A (20 mM Tris-HCl, pH 8.0, 100 mM NaCl). The GST-plectin1-339 fusion protein was generated and immobilized on glutathione-Sepharose beads as described previously (Geerts et al., 1999). The beads were incubated in buffer A containing 1% (w/v) BSA to block non-specific binding sites. After washing, 750 pmoles of GST-plectin1-339 beads were added to the diluted cell lysates and the mixtures were incubated overnight at 4°C. The Sepharose beads were washed once with buffer A and then sedimented through a sucrose cushion (800 mM sucrose in buffer A). Finally, beads were boiled in SDS-sample buffer and bead-associated proteins were separated by SDS-PAGE and identified by immunoblotting using mAb 12CA5 against HA.
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RESULTS |
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The unequal distribution of the ABS sequences within the CH1 and CH2 domains and the differences in their sequences (Fig. 1A) raise the possibility that these two CH domains have different functions. In addition, recent studies indicate that the single CH domain of the calponin protein is not necessary for F-actin binding, but that sequences located near the CH domain have a role in its interaction with actin (Gimona and Mital, 1998; Corrado et al., 1994). This prompted us to map the plectin sequences involved in actin binding and to gain more insight into the roles of the plectin CH and ABS sequences in the interaction with actin.
Yeast two-hybrid assays for interactions of plectin with actin were performed by cotransformation of the yeast strain PJ69-4A with pACT2-derived plasmids encoding the transcriptional activation domain (AD) of Gal4 fused to full-length actin, together with pAS2-1-derived plasmids encoding fusions between the DNA-binding domain (DB) of Gal4 and different fragments of the plectin-ABD (Fig. 1B). Expression of the fusion proteins was confirmed by immunoblotting with anti-Gal4(BD) or anti-Gal4(AD) antibodies (not shown). Interactions of plectin with actin were detected by the growth of yeast colonies on selective SC-LTHA plates (see Materials and Methods). A high plating efficiency was observed when plectin1-339 was coexpressed in yeast with full-length ß- or -cytoplasmic actin, or
-actin from skeletal muscle, showing that the His and Ade reporter genes were efficiently expressed as a result of a strong interaction between plectin1-339 and the different actin isoforms. Plectin1-339 contains unique sequences that are not part of the plectin-ABD (amino acids 1-65, encoded by exon 1c, and amino acids 302-343, encoded by exon 9 of plectin). To localize the region within the ABD domain that interacts with actin and to exclude the possibility that sequences outside of the plectin-ABD are also involved, a series of plectin deletion constructs were tested. C-terminal truncation of amino acids 237-339 does not abolish interaction, but in contrast results in a moderate increase in the binding of the plectin-ABD to all the actin isoforms tested. Further truncation extending to the C-terminal end of the first CH domain (residues 173-236) also does not alter binding, showing that the plectin CH2 domain is not necessary for actin binding (Fig. 1B). In contrast, deletion of the second ABS completely abolished the interaction of plectin with
-, ß- and
-actin, showing that this region is required for binding to ß-and
-cytoplasmic actin, as well as to
-skeletal muscle actin.
Does plectin-ABD bind to G-actin and F-actin filaments?
Although plectin decorates actin stress fibers in cultured cells, suggesting that plectin interacts with polymeric actin, no evidence for direct binding has yet been presented. To test whether the plectin-ABD mediates direct interactions with actin filaments, copolymerization assays were performed using purified monomeric -actin from skeletal muscle and an MBP-fusion protein containing the first 339 amino acids of human plectin. Prior to the induction of polymerization, monomeric actin at 2.5 µM and MBP-plectin1-339 in actin G buffer were mixed at the molar ratio indicated in Fig. 2A. Actin polymerization was induced by the addition of initiation mix and actin filaments were sedimented by centrifugation. As shown in Fig. 2A, MBP-plectin1-339 was found associated with the actin filaments recovered in the pellet. Quantification by scanning densitometry of Coomassie Brilliant Blue-stained gels revealed that plectin has no influence on the amount of pelleted actin, thus indicating that MBP-plectin1-339 neither promotes nor inhibits actin polymerization (not shown and see below). Scatchard analysis revealed that MBP-plectin1-339 binds to actin with a Kd of 0.3 µM and a molecular ratio of 1:1 (Fig. 2B). These estimations were made by assuming that the plectin molecules that remain in the supernatant were not associated with unpolymerized actin: at a low plectin concentration (lane 1) less than 5% of plectin is found in the supernatant although it contains unpolymerized actin. Similar experiments performed with a GST-fusion protein containing the first 339 amino acids of the plectin N terminus, GST-plectin1-339, gave similar results (data not shown).
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Effects mediated by plectin-ABD on the kinetics of actin polymerization
Using two different assays, pyrene-actin fluorescence and light scattering, we next investigated whether plectin can modulate the actin polymerization rate by binding to F-actin. In the pyrene-actin assay the fluorescence signal is directly proportional to the degree of actin polymerization. However, as this test relies on the fact that F-actin is more fluorescent than G-actin because the environment of the labeled residue (Cys374) is different in the G and F-actin molecules, an actin ligand that alters the three-dimensional structure of F-actin would also affect fluorescence. On the contrary, light scattering is influenced by the length and bundling of filaments. Although probably less reliable than the pyrene-actin assay, this test has the advantage that unmodified actin is used and that it does not rely on a conformational change of actin.
Testing the effect of plectin on the rate of actin polymerization in the pyrene-actin assay revealed that MBP-plectin1-339 increases the rate of actin polymerization in a concentration-dependent manner (Fig. 3A). No differences between the steady-state concentrations of F-actin in the absence or presence of plectin-ABD were detectable by this assay at the concentrations tested (not shown). Thus, the plectin-ABD does not alter the degree of actin polymerization, which is in agreement with the copolymerization results. As the pyrene-actin assay measures spontaneous polymerization, we conclude that the effects of the plectin-ABD on the rate of fluorescence likely result from an increase in the number of actin nuclei, which increase the amount of polymerizing actin and therefore the fluorescence.
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The data obtained by light scattering in combination with the nucleating effect of the plectin-ABD observed using the pyrene-actin assay led us to assume that MBP-plectin1-339 can probably bundle actin filaments by cross-linking at least two actin molecules.
Evidence for a bundling activity mediated by plectin-ABD dimerization
Cross-linking and/or bundling of actin was visualized by electron microscopy of the polymerization mixtures monitored by light scattering. In the absence of plectin, long actin filaments were randomly distributed all over the grid with only some of them organized into bundles (Fig. 4A). At a concentration of 0.5 µM, MBP-plectin1-339 notably increased the number of actin bundles (Fig. 4B). These bundles consisted of 4-6 actin filaments in juxtaposition and were partially organized in a branched network. Due to the presence of globular particles, corresponding to the plectin-fusion protein, the bundles have a rather rough appearance. The globular particles were found regularly distributed at 30 nm intervals along the filaments. To confirm that plectin indeed induces bundling of actin filaments, low-speed sedimentation assays were performed using MBP plectin1-339 and MBP alone as a control.
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Identification of the dimerization domain of plectin-ABD
The role of the CH domains in actin binding remains controversial (Gimona and Mital, 1998). There is stronger homology between the CH1 and CH2 domains, respectively, of different actin-binding proteins (Fig. 1A; Van Troys et al., 1999) than between CH1 and CH2 of the same protein, supporting the current idea that although CH1 and CH2 domains are structurally related, their function is most probably distinct (Van Troys et al., 1999). Here, we have found that the CH1 domain of plectin is necessary for actin binding (see above), while CH2 is not. Altogether these data and the observed bundling mediated by plectin-plectin interactions prompted us to investigate which part of the plectin N terminus is required for this interaction. To this end, full-length and truncated plectin-ABD were tested in a yeast two-hybrid assay and the resulting binding activities were compared to the binding obtained with ß-actin (Fig. 6). We observed that plectin1-339 (as a Gal4(BD)-fusion protein) interacted with both plectin1-339 (as a Gal4(AD)-fusion) and ß-actin. In addition, the plectin mutant in which the 35 N-terminal amino acids had been deleted (plectin36-339) still bound to other plectin molecules as well as to ß-actin. An N-terminal deletion that removed all 64 amino acids encoded by exon 1c completely abolished the interaction with ß-actin, without altering the interaction with other plectin molecules. However, by also deleting residues 65-173 or 65-284, the binding to other plectin1-339 molecules was also abolished. Since plectin36-339 is able to bind to both ß-actin and plectin1-339, we tested the effect of C-terminal truncations of this molecule on these interactions. Removal of the CH2 domain enhances binding to ß-actin but does not alter binding to plectin1-339, whereas deletion of ABS2 abrogates binding to both ß-actin and plectin1-339. These findings indicate that for dimerization at least one intact CH1 domain in any protein of the dimer is required.
To determine whether the plectin CH1 domain alone can support dimerization of the plectin-ABD we performed a similar yeast two-hybrid analysis using a Gal4(AD)-fusion protein containing amino acids 36-181 of plectin. Consistent with the results described above we found that plectin36-181 interacts with plectin1-339 (Fig. 6) with an efficiency similar to that of plectin1-339. In contrast to plectin1-339, plectin36-181 was unable to interact with plectin65-339. This latter finding suggests that sequences in the region of residues 36-65 may also contribute to plectin-ABD binding. Alternatively, these sequences may be required to position the CH1 domain at a sufficient distance from the Gal4 moiety to make binding possible in yeast. In agreement with the results obtained with plectin1-339, we found that deletion of ABS2 (plectin1-142, plectin5-142, plectin36-142) completely abolished plectin36-181-binding activity.
Can other ABDs mediate homo- and heterodimerization?
Based on the strong homology in sequences between the ABDs of the ß-spectrin family members, we speculate that ABDs other than those of plectin may homodimerize and even heterodimerize. This assumption was tested by yeast two-hybrid assays using Gal4-fusion proteins containing the N-terminal ABDs of dystrophin, dystonin and plectin. We found that plectin1-339 binds to other plectin1-339 molecules and to dystonin-21-336 with similar efficiencies (Fig. 7). By contrast, there was no binding between plectin1-339 and dystrophin1-337 or between dystonin-21-336 and dystrophin1-337. Dystonin-21-336 can bind to other dystonin-21-336 molecules but this binding appeared to be weaker than that with plectin1-339. Binding of dystrophin1-337 to other dystrophin1-337 molecules proved to be very weak and was barely detectable. Since in the Gal4-fusion protein, the dystrophin-ABD, which starts at residue 10, is located closely to the Gal4 protein, interaction between two of these fusion proteins via their dystrophin-ABDs may not be possible because of steric hindrance by the Gal4-moiety. We therefore tested whether the efficiency of intermolecular binding between two dystrophin-ABDs could be made possible by increasing the size of the linker region between the ABD and the Gal4-moiety. To achieve this, we created a chimeric protein in which the first 65 residues of plectin separated the Gal4-moiety and the dystrophin-ABD. Testing this chimeric protein in the yeast two-hybrid assay indeed revealed efficient intermolecular binding of the chimeric proteins and a somewhat reduced binding of it to dystrophin1-337. There were however, still no interactions with either plectin1-339 or dystonin-21-336.
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DISCUSSION |
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The actin-binding site in the plectin-ABD is contained within the first CH domain, since truncation at either the N-terminal (Fig. 6) or the C-terminal end of CH1 (Fig. 1B) abolished the binding of plectin to the different actin isoforms. Our yeast two-hybrid assays clearly show that residues 143-172, comprising the ABS2, are required for mediating binding to all the actin isoforms tested. Recently, analyses of the three-dimensional structure of fimbrin and ß-spectrin ABDs suggested that the major actin-binding site is localized in the last -helix of CH1, corresponding to a sequence containing amino acid residues of the ABS2 (Banuelos et al., 1998). Dystrophin, another member of the ß-spectrin family, also binds to actin by a conserved ABD. However, in this latter case binding requires both the ABS1 and the ABS2 (Fabbrizio et al., 1993; Corrado et al., 1994). The crystal structure of the first CH domain of human ß-spectrin (MMDB Id: 8495) (Carugo et al., 1997) indicates that the first
-helix containing the ABS1 is located in close proximity to the last
-helix, which contains the ABS2. Thus, amino acid residues located in the ABS1 might also participate in actin binding. The observed inability of the complete plectin-ABD fragment to interact with actin (this study and unpublished observations) might be due to steric hindrance of the ABS2 resulting from the fusion to Gal4(BD) and GST. The same mechanism might account for the different results obtained by yeast two-hybrid and cell transfection experiments regarding the involvement of residues 36-65 of plectin in actin-binding. Indeed, we found that fusion proteins (Gal4 or GST) containing plectin65-339 were unable to bind actin in yeast two-hybrid and in vitro interaction assays. However, the colocalization of actin and plectin65-339 identified in transfected REF cells strongly suggests that the first 64 amino acid residues are not necessary for actin binding.
The results of our study indicate that the plectin CH2 domain is not critically implicated in the binding to actin. However, it appears to have a regulatory function on actin binding since its partial or complete removal increases plectin-actin binding. It is possible that the partial inhibition mediated by the CH2 domain results from hindrance of sequences involved in plectin-actin interactions and thus proteins that interact with the CH2 could potentially modulate the interaction of the plectin-ABD with actin.
In cosedimentation assays, we found that plectin binds to actin with an apparent Kd of 0.3 µM and in a molecular ratio of 1:1. These properties are similar to those of other actin-binding proteins containing the conserved ABD such as dystrophin, -actinin and dystonin (for a review, see Hartwig, 1994). In addition, in vitro experiments have shown that the plectin-ABD does not modify the amount of polymerized actin, suggesting that the plectin-ABD does not prevent polymerization of G-actin into F-actin. Consistent with this finding, pull-down assays, performed with immobilized plectin-ABD, demonstrate that plectin hardly binds to G-actin. These data are in apparent contrast to those obtained by Andrä et al. (Andrä et al., 1998), who showed that the plectin-ABD binds to monomeric actin using ELISA assays. These authors reported a Kd of 0.32 µM, the same as we established for F-actin. The discordance concerning the binding of plectin to G-actin is probably because Andrä and coworkers used a buffer containing 2 mM Mg2+, with ionic conditions that favor the formation of F-actin. Thus, like other ß-spectrin family members (i.e.
-actinin, dystrophin), plectin appears to bind to F-actin with a much higher affinity than to G-actin. Although the yeast two-hybrid assays are normally based on interactions between two monomeric Gal4-fusion proteins, oligomerization of one of the two partners has ocassionally been observed when other cytoskeleton proteins were studied (Meng et al., 1996), suggesting that short F-actin structures can probably be assembled in the yeast nucleus, thereby providing an appropriate ligand for Gal4-plectin-ABD.
The proteins of the ß-spectrin family are well-established actin cross-linkers. This property is attributed to protein dimerization mediated by their rod domain. However, recent crystallographic studies showed that in a crystal the ABDs of utrophin and dystrophin are organized in a dimer (Keep et al., 1999; Norwood et al., 2000), which suggests that the ABD of these proteins can also participate in the process of dimerization. Consistent with these findings, we demonstrate that the ABD of plectin is able to form dimers and thereby to bundle actin filaments in vitro. In addition, using pyrene-actin assays, we show that the plectin-ABD increases the rate of actin polymerization and reduces the lag phase, probably by nucleating actin filaments. We assume that this nucleation of actin filaments by the plectin-ABD is also mediated by its dimerization, which might stabilize actin oligomers or even induce their formation. However, we cannot exclude that the plectin-ABD modulates actin polymerization by increasing the rate of monomer addition to barbed ends or by severing actin filaments, thereby creating new barbed ends. By employing the yeast two-hybrid system we have mapped the dimerization domain of the plectin-ABD in the CH1 domain. Thus, the dimerization of the plectin-ABD is different from that of the utrophin-ABD, since the dimerization of the latter is thought to be mediated by CH1-CH2 interaction (Keep et al., 1999). Unexpectedly, we found that the ABS2 is required for both actin binding and for plectin-plectin interaction. Nevertheless, the residues involved in binding are probably different, since the plectin-ABD65-339 construct can associate with the plectin-ABD1-339 but lacks actin-binding properties. These observations suggest that dimerization of the plectin-ABD does not prevent actin binding, which is supported by our cosedimentation assays showing that at low concentrations, all of the plectin-ABDs are bound to F-actin. Also, if plectin dimerization inhibited actin binding, then actin-plectin interaction would not have been detected by either the yeast two-hybrid or in vitro binding assays. The distinct binding activities might be explained by the presence of an actin-binding site and a dimerization site located on opposite sides of the first plectin CH domain. Supporting this assumption, Correia et al. (Correia et al., 1999) have shown that the CH1 of fimbrin mediates interactions with actin and vimentin. Using three-dimensional reconstruction a model was proposed by these authors in which actin and vimentin binding sites are located at two opposite sides in the CH1 domain of fimbrin. Besides its role in the bundling of actin filaments, dimerization of the plectin-ABD could also be essential for the binding of actin itself. This is supported by the results of the yeast two-hybrid assays, showing that sequences required for actin binding overlapped with those required for the dimerization of the plectin-ABD. Although the results of the yeast two-hybrid assay suggest a role of the plectin residues 36-65 in dimerization, we assume that, as for binding of plectin to actin, the absence of binding between plectin36-181and plectin65-339 is due to steric hindrance mediated by the Gal4-moiety in plectin65-339.
The in vitro formation of plectin dimers and oligomers has been described (Foisner and Wiche, 1987), and it is believed that they are formed via the rod domain (for reviews, see Wiche, 1989; Foisner and Wiche, 1991). However, while the formation of dimers might be mediated via coiled-coil interactions of the rod domains, oligomerization is likely to require additional binding sites. Our study strongly indicates that the plectin-ABD could provide such additional binding sites. Although it is not yet clear whether the dimerization of the plectin-ABD has physiological relevance, there is evidence that plectin by itself can assemble into a filamentous network in cultured cells (Wiche and Baker, 1982; Wiche et al., 1984).
Using a yeast two-hybrid assay we extended our findings on the dimerization of the plectin-ABD to other ß-spectrin family members. We found that dystonin and dystrophin ABDs can also dimerize, but less efficiently than the plectin-ABD. In the case of dystrophin, efficient binding was only obtained with a chimeric molecule containing the first 65 amino acid residues of plectin. Using the same approach we identified the ABD of plectin as a ligand for the dystonin-ABD, a neuronal isoform of the bullous pemphigoid antigen 1 protein (Ruhrberg and Watt, 1997), which strongly suggests that ABDs of different proteins can form heterodimers. By contrast no clear interaction was identified between either the plectin or the dystonin-ABDs with that of dystrophin, which indicates that ABD heterodimerization is specific. The basis of this specificity is probably determined by differences in the sequences, as dystonin and plectin display 88% of homology in their ABD, whereas the homology is 61% in the case of the ABDs of plectin and dystrophin. This heterodimerization of plectin with dystonin further reinforces the role of plectin as a multifunctional cytolinker protein and creates new possibilities for investigating its importance in the regulation of actin dynamics in the cell.
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ACKNOWLEDGMENTS |
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