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The tumor suppressor Scrib selectively interacts with specific members of the zyxin family of proteins
FEBS 29904
FEBS Letters 579 (2005) 5061–5068
The tumor suppressor Scrib selectively interacts with specific members of the zyxin family of proteins Marleen M.R. Petit, Koen R.M.O. Crombez, Hilke B.V.K. Vervenne, Nancy Weyns, Wim J.M. Van de Ven* Laboratory for Molecular Oncology, Department of Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology (VIB), Herestraat 49, B-3000 Leuven, Belgium Received 12 January 2005; revised 7 July 2005; accepted 9 August 2005 Available online 19 August 2005 Edited by Berend Wieringa
Abstract The zyxin family of proteins consists of five members, ajuba, LIMD1, LPP, TRIP6 and zyxin, which localize at cell adhesion sites and shuttle to the nucleus. Previously, we established that LPP interacts with the tumor suppressor Scrib, a member of the leucine-rich repeat and PDZ (LAP) family of proteins. Here, we demonstrate that Scrib also interacts with TRIP6, but not with zyxin, ajuba, or LIMD1. We show that TRIP6 directly binds to the third PDZ domain of Scrib via its carboxy-terminus. Both proteins localize in cell–cell contacts but are not responsible to target each other to these structures. In the course of our experiments, we also characterized the nuclear export signal of human TRIP6, and show that LIMD1 is localized in focal adhesions. The binding between two of zyxinÕs family members and Scrib links Scrib to a communication pathway between cell–cell contacts and the nucleus, and implicates these zyxin family members in Scrib-associated functions. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Lano; Erbin; Densin-180; Let-413; Scribble; KIAA0147
1. Introduction At sites of cell adhesion, an increasing number of proteins not only performs structural tasks, such as sustaining cell shape, but is also involved in signaling events. A family of proteins that may operate as such is the zyxin family of LIM domain proteins. Members of this family localize at sites of cell adhesion, such as focal contacts, which are membrane attachment sites to the extracellular matrix, and cell–cell contacts. However, apart from this localization in cell adhesion sites, these proteins also transiently localize in the nucleus [1–5]. The zyxin family consists of five members: zyxin [6], TRIP6 (thyroid receptor interacting protein 6) [7], LPP (lipoma pre* Corresponding author. Fax: +32 16 34 60 73. E-mail address: [email protected] (W.J.M. Van de Ven).
Abbreviations: GFP, green fluorescent protein; GST, glutathione S-transferase; LAP, leucine-rich repeat and PDZ; LIMD1, LIM domain containing 1; LPP, lipoma preferred partner; LRR, leucine-rich repeat; NES, nuclear export signal; PBS, phosphate buffered saline; PDZ, PSD95/discs-large/ZO-1; TRIP6, thyroid receptor interacting protein 6
ferred partner) [8], ajuba [9] and LIMD1 (LIM domain containing 1) [10]. A recent study of the Drosophila genome sequence indicates that zyxin, LPP, and TRIP6 form one paralogous group in vertebrates that is represented by the zyx102 protein in the fruit fly, while ajuba and LIMD1 form another group that is represented by the CG11063 protein (Fig. 1) [11]. All family members share a similar organization having three LIM domains (zinc-finger protein interaction domains) at their carboxy-terminus, which is preceded by a proline-rich preLIM region containing a number of protein interaction domains. While all family members have a high degree of amino acid similarity in their LIM domains, their pre-LIM regions are quite divergent. Because of their structural features and their characteristic to shuttle between the nucleus and the cytoplasm, zyxin family members have been proposed to be scaffolding proteins involved in signal transduction from sites of cell adhesion to the nucleus. In a previous study, we identified the tumor suppressor Scrib as an interaction partner of one of zyxinÕs family members, LPP [12]. Scrib is a member of the LAP (LRR (leucine-rich repeat) and PDZ (PSD-95/discs-large/ZO-1)) family of membrane-associated proteins [13], which consists of four members: Densin-180, Erbin, Lano, and Scrib [14–17]. LAP proteins contain a set of LRRs at their amino-terminus, and four (Scrib), one (Erbin, Densin-180) or no (Lano) copies of the PDZ domain. A specific characteristic of these proteins are the LAP-specific domains (LAPSa and b), which are located carboxy-terminally of the LRRs [18]. LPP binds to the PDZ domains of Scrib via its C-terminus. Little information is available at the moment about the function of Scrib. Relating to the control of cell polarity and proliferation, Scrib is a functional homologue of the Drosophila scribble protein [19]. Polarity defects [20] and tumorous overgrowth in Scribblemutant flies [21] are rescued by Scrib predicting an important role for Scrib in the suppression of mammalian tumorigenesis. This hypothesis is further supported by the finding that highrisk papilloma virus E6 proteins direct Scrib for degradation by directly binding to the PDZ-domains of Scrib [17,22]. Here, we report about the differential interaction between Scrib and members of the zyxin family. We found that Scrib, besides with LPP, also selectively interacts with TRIP6, but not with zyxin, ajuba or LIMD1. The binding between Scrib and TRIP6 is mediated by the third PDZ domain of Scrib and the C-terminus of TRIP6. Furthermore, we have studied whether Scrib is involved in the subcellular targeting of TRIP6 to focal adhesions and to cell–cell contacts.
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.08.012
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M.M.R. Petit et al. / FEBS Letters 579 (2005) 5061–5068 containing all four PDZ domains) (wild-type or mutated) in NENT100 buffer (100 mM NaCl, 20 mM Tris–HCl, pH 7.6, 1 mM EDTA, 0.1% NP-40, protease inhibitors). This mixture was tumbled overnight at 4 C. Subsequently, the beads were washed 5 times in 500 ll NENT100 buffer, resuspended in 25 ll SDS–PAGE sample buffer. Proteins were separated by SDS–PAGE and interacting Scrib was detected by autoradiography.
Fig. 1. Schematic phylogenetic representation of zyxin family members with their C-termini and requirements for binding to Scrib-type PDZ domains. h, hydrophobic amino acid.
2. Materials and methods 2.1. Plasmid constructs Constructs expressing wild-type or mutated human zyxin, TRIP6, ajuba, and LIMD1 as green fluorescent protein (GFP) fusions were made by cloning the coding regions in the pEGFP-C vectors (Clontech). TRIP6 en zyxin cDNAs, and ajuba and LIMD1 cDNAs were kindly provided by Mary Beckerle (University of Utah, USA) and Greg Longmore (Washington University School of Medicine, USA), respectively. The constructs expressing Xpress-hScrib-mito and Xpress-hScribdPDZ-mito were described before [12]. Mutations were made using the QuikChangee site-directed Mutagenesis Kit (Stratagene). All synthetic mutations, ligation sites and PCR-amplified regions were verified by sequencing. 2.2. Cell culture, stable cell lines and transfections Cell lines included CV-1 (ATCC CCL-70), HEK293 (ATCC CRL1573), 293T (HEK 293 cells expressing SV40 T-antigen), and MDCK strain II (Dog normal kidney epithelial cells). Cells were grown in DMEM/F12 (1:1) (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and cultured at 37 C in a humidified CO2 incubator. Transient transfections were performed using FuGenee 6 Transfection Reagent (Roche). Stable MDCKII cell lines were made expressing wild-type and carboxy-terminally mutated human GFP-TRIP6 proteins. Transfection of MDCK cells was performed using Lipofectamine 2000 Reagent (Life Technologies). Transfected cells were selected in medium containing 250 lg/ml G418 (Life Technologies), and resistant colonies were isolated 10–14 days later. Individual clones were screened for expression of the GFP fusion proteins by Western blotting using a rabbit polyclonal anti-GFP antibody (Tebu Bio). 2.3. Mammalian two-hybrid system Bait- and prey-constructs were made using pM-vectors [23] and the pSNATCH-vector [24], respectively. 24 h upon seeding, semi-confluent HEK293 cells on 24-well plates were transiently cotransfected with 100 ng bait-DNA, 100 ng prey-DNA, 250 ng DNA of a luciferase reporter construct [12] and 50 ng of CMV-b-galactosidase DNA (internal control for transfection efficiency). Cell lysates were prepared 18–24 h after transfection and assayed for luciferase activity as described previously [1]. 2.4. In vitro transcription/translation and GST pull-down assays All in vitro translation reactions were carried out using the TNT T7 Quick Coupled Transcription/Translation System (Promega). For GST pull-down assays, bacterial expression constructs were made using pGEX-5X vectors (Amersham-Pharmacia Biotech) directing the synthesis of glutathione S-transferase (GST) fusion proteins containing wild-type or mutated forms of human TRIP6. These fusion proteins were purified according to manufacturerÕs instructions and verified by SDS–PAGE. GST fusion proteins or GST alone, bound to glutathione–agarose beads, were incubated with in vitro synthesized [35S] methionine-labeled human Scrib protein (amino acids 616–1490
2.5. GFP-fluorescence and indirect immunocytochemistry CV-1 or 293T cells seeded on glass coverslips, and MDCKII cells seeded on Transwell-Clear polyester membranes (0.4 lm, Costar) were fixed in 4% formaldehyde for 20 min at room temperature followed by three washes in phosphate buffered saline (PBS) containing 0.1 mM CaCl2 and 0.1 mM MgCl2 (PBS++). For GFP-fluorescence, slides were mounted in vectashield mounting medium (Vector Laboratories, Inc.) and analyzed on a Zeiss Axiophot fluorescence microscope equipped with an RT slider SPOT camera (Diagnostic Instruments, Inc.) using SPOT RT Software v3.4, or by confocal microscopy (MRC-1024 Laser Scanning Confocal Imaging System, Bio-Rad). Indirect immunocytochemistry was done as described [12]. Primary antibodies: a rabbit polyclonal anti-GAL4 DNA-binding domain antibody (Tebu Bio), a monoclonal anti-Xpress antibody (Life Technologies), and a monoclonal anti-vinculin antibody hVIN-1 (Sigma). Secondary antibodies: fluorescently-tagged Alexa antibodies (Molecular Probes).
3. Results 3.1. Scrib binds to the C-terminus of TRIP6 via its PDZ domains In a previous study [12], we identified Scrib as an interaction partner of LPP, which is a member of the zyxin family of proteins. The binding between Scrib and LPP is mediated via the PDZ domains of Scrib and the C-terminus of LPP. The binding specificity of PDZ domains to C-termini, which is the typical mode of interaction of PDZ domains, is critically determined by the interaction of the residue at position aB1 and the side chain of the 2 residue of the C-terminal ligand [25,26]. Since all four PDZ domains of Scrib contain a histidine at position aB1, the carboxy-terminal sequence of Scrib target proteins is predicted to require a hydrophobic amino acid (h) at the 0 (carboxy-terminus) position, and a serine (S) or threonine (T) at the 2 position (Fig. 1). When comparing the carboxy-terminal tails of all human zyxin family members, we noticed that, besides LPP, also the carboxy-terminus of TRIP6 theoretically fulfils the criteria for binding to the PDZ domains of Scrib, while the C-termini of zyxin, ajuba and LIMD1 do not (Fig. 1). This means that, at least in theory, Scrib would be able to bind to the C-terminus of TRIP6 but not to the C-terminus of zyxin, ajuba or LIMD1. To evaluate these predictions experimentally, we first performed a mammalian twohybrid experiment. We used as baits a C-terminal portion of all zyxin family members, consisting of the LIM-domains and the C-terminal tail of the respective proteins. These baits were designated as follows: pM-TRIP6LT, pM-zyxinLT, pMajubaLT and pM-LIMD1LT. As prey-protein, we used pSNATCH-hScribPDZ containing a part of the human Scrib protein (amino acids 669–1233) surrounding all four PDZ-domains. As summarized in Fig. 2A, the interaction between TRIP6 and Scrib resulted in high levels of luciferase reporter activity. These high levels were not obtained when using the baits containing zyxin, ajuba, or LIMD1. These results suggest that, besides with LPP, Scrib also interacts with TRIP6 but not with zyxin, ajuba or LIMD1.
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Fig. 2. Scrib binds to the C-terminus of TRIP6 via its PDZ domains. pM-bait- and pSNATCH-prey-constructs were cotransfected into 293 cells in the combination indicated (LT: LIM domains and carboxyterminal tail), together with a GAL4-regulated luciferase reporter and a CMV-b-galactosidase internal control. Cell lysates were assayed for luciferase activity 18–24 h after transfection. Relative luciferase activity is reported as the average of three independent duplo experiments (with standard error).
Interaction between bait- and prey-proteins in a mammalian two-hybrid assay takes place in the nucleus. For an accurate performance of this assay, this means that bait- and preyproteins should be accumulating in the nucleus. In the abovedescribed mammalian two-hybrid experiment, where we used partial bait proteins, all baits, also the ones of zyxin, ajuba and LIMD1, were well expressed on Western blot and were accumulating in the nucleus of the cells (results not shown). In a follow-up experiment, we wanted to confirm the interaction between TRIP6 and Scrib, and to determine the amino acids in TRIP6 that are critical for its interaction with the PDZ domains of TRIP6, however, this time using the full length TRIP6 protein as a bait. Since TRIP6 contains a nuclear export signal (NES) in its pre-LIM region, we had to use bait-proteins in which this NES had been inactivated. However, for TRIP6, a NES is only characterized for the mouse protein [3]. As it was reported for zyxin that it has one or two NESs depending on the species [27], we wanted to evaluate first whether the NES that had been mapped in the mouse TRIP6 protein was also the NES of the human protein. In mouse TRIP6, three leucine residues are critical for nuclear export (Fig. 3A). When these residues are mutated into alanine residues, nuclear export of mouse TRIP6 is abolished
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[3]. We mutated the corresponding leucine residues in the human TRIP6 protein into alanine residues in a GFP-hTRIP6WT fusion (Fig. 3A). Wild-type and mutated proteins were introduced into CV-1 fibroblast cells. While GFP-TRIP6WT, containing full length wild-type human TRIP6 with an intact NES, was excluded from the nucleus (Fig. 3B, left panel), GFP-TRIP6dNES, containing full length human TRIP6 with the above-described leucine-to-alanine mutations, was accumulating in the nucleus (Fig. 3B, right panel). To verify whether mutation of the NES in human TRIP6 also induced nuclear accumulation of the bait-protein that was used in the mammalian two-hybrid assay, we introduced wild-type and mutated TRIP6-bait-proteins in 293T cells. While pMTRIP6WT, containing GAL4-fused full length wild-type human TRIP6 with an intact NES, was excluded from nuclei (Fig. 3C, upper panels), pM-TRIP6dNES, containing GAL4fused full length human TRIP6 with the above-described leucine-to-alanine mutations, was accumulating in the nuclei of the cells (Fig. 3C, lower panels). These results indicate that, not only in mouse but also in human TRIP6, the leucines at positions 100, 104 and 107 are critical for proper nuclear export of the protein. To perform the follow-up two-hybrid experiment, we used as baits for TRIP6, pM-TRIP6dNES, and pM-TRIP6dNES,T474A and pM-TRIP6dNES,C476A, which are identical to pMTRIP6dNES except for a point mutation to alanine introduced at threonine474 (position 2) and cysteine476 (position 0), respectively, the two residues that are theoretically critical for interaction with the Scrib PDZ domains. pSNATCHhScribPDZ was again used as prey. As summarized in Fig. 2B, the interaction between wild-type full length TRIP6 and the Scrib PDZ domains resulted in high levels of luciferase reporter activity. These high levels dropped to background when pM-TRIP6dNES,T474A or pM-TRIP6dNES,C476A were used as baits. The ‘‘background’’ levels of luciferase that were detected when pM-TRIP6-baits were used in combination with pSNATCH (empty prey-vector) as prey, are due to the intrinsic transcriptional activation activity of the human TRIP6 protein, as has been shown for mouse TRIP6 [3]. These results indicate that TRIP6 binds to the PDZ domains of Scrib and that amino acids at position 0 and 2 are critical for this interaction. 3.2. Scrib interacts with LPP and TRIP6 in vivo but not with zyxin, ajuba or LIMD1 Evidence for an in vivo interaction between Scrib and TRIP6 was obtained by performing mitochondrial targeting experiments. We tested if Scrib was sufficient to recruit TRIP6 to an ectopic location in vivo. The membrane anchor of the ActA sequence has been shown previously to be sufficient to target proteins expressed in mammalian cells to the surface of mitochondria [28,29]. This ectopic localization allows testing ligand recruitment in vivo. For this purpose, we generated a chimera named Xpress-hScrib-mito made up by an Xpress-epitope tag fused to the amino-terminus of human full length Scrib and linked in frame to the membrane anchor of the Listeria monocytogenes protein ActA (mito). CV-1 cells were transiently transfected with Xpress-hScribmito and full length wild-type or carboxy-terminally mutated TRIP6 GFP-fusions. Cells were labeled with an anti-Xpress antibody and examined by fluorescence microscopy. As shown in Fig. 4A, the Xpress-hScrib-mito chimera localized
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Fig. 3. Mapping of the NES in human TRIP6. (A) Alignment of a portion of the LPP amino acid sequence with the corresponding sequence in mouse and human TRIP6. The boxed region in LPP and the boxed leucine residues in mouse TRIP6 were previously shown to be essential for nuclear export of the respective proteins [1,3]. The boxed leucine residues in human TRIP6 were mutated into alanines in the experiments presented here, and shown to be part of a unique NES in the human TRIP6 protein. (B) CV-1 fibroblasts were transiently transfected with GFP-hTRIP6 proteins as indicated. GFP fusions were visualized by epifluorescence microscopy. A focal plane corresponding to the cell body is shown. Insets are focused on focal adhesions. (C) 293T epithelial cells were transiently transfected with pM-hTRIP6 proteins as indicated. pM-hTRIP6 fusions were stained with anti-GAL4 antibodies and visualized by epifluorescence microscopy. Nuclei were visualized by staining with DAPI.
to mitochondria. While wild-type TRIP6 was recruited to Xpress-hScrib-mito on mitochondria, this recruitment was abolished when the carboxy-terminus of TRIP6 was mutated (Fig. 4A). To investigate the importance of the PDZ domains of Scrib in this recruitment of TRIP6, we deleted all four PDZ domains (amino acids 724-1192) from Xpress-hScrib-mito (= Xpress-hScribdPDZ-mito), and tested whether this PDZless protein still was able to recruit TRIP6 to mitochondria. As shown in Fig. 4A, lower panels, Xpress-hScribdPDZ-mito lost its ability to recruit TRIP6 to mitochondria. These results indicate that the PDZ domains of Scrib are an absolute requirement for its binding to TRIP6. We next investigated whether the remaining family members of LPP and TRIP6 could be recruited to Xpress-Scrib-mitocoated mitochondria. CV-1 cells were cotransfected with Xpress-hScrib-mito and full length human zyxin, ajuba, and LIMD1 GFP-fusions respectively. As shown in Fig. 4B, no recruitment could be detected. Interesting to note here is that we could easily detect GFP-LIMD1 in, what looked like, focal adhesions. To find out whether GFP-LIMD1 indeed localizes to focal adhesions, we performed double labeling experiments. CV-1 cells were transfected with GFP-LIMD1, and labeled with vinculin antibodies used as a marker for focal adhesions. As shown in Fig. 5, GFP-LIMD1 indeed colocalized with vin-
culin in focal adhesions. To our knowledge, this is a novel finding, and taken together with what has been published for zyxin [6], TRIP6 [30], LPP [1], and ajuba [31,32], this indicates that all of zyxinÕs family members can localize in focal adhesions. In conclusion, our results indicate that Scrib selectively binds to two of zyxinÕs family members, being LPP and TRIP6, and not to zyxin, ajuba, and LIMD1. 3.3. Direct interaction between the carboxy-terminus of TRIP6 and the PDZ domains of Scrib To demonstrate the direct interaction between TRIP6 and Scrib-PDZ domains, we performed GST pull-down experiments. In vitro translated Scrib-PDZ domains were tested for binding with glutathione beads, which were coupled with GST-TRIP6-LTWT, GST-TRIP6-LTC476A, or GST alone. GST-TRIP6-LTWT contains the three LIM domains and the wild-type carboxy-terminal tail of human TRIP6. GSTTRIP6-LTC476A is identical to GST-TRIP6-LTWT except for a point mutation to alanine introduced at cysteine476 (position 0). All GST-fusion proteins as well as GST alone were expressed well in Escherichia coli (Fig. 6A). As shown in Fig. 6B, ScribPDZ domains interacted specifically with the wild-type TRIP6 protein but not with its mutated form, GST-TRIP6-LTC476A, or with GST alone. These results indicate that there is a specific and direct interaction between TRIP6 and Scrib.
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Fig. 5. LIMD1 localizes in focal adhesions. CV-1 cells were transiently transfected with GFP-LIMD1, and stained with an anti-vinculin antibody to detect endogenous vinculin. GFP fluorescence (left panel) and vinculin staining (right panel) were visualized by epifluorescence microscopy.
binding loop (LG fi AE), and tested how efficiently these mutated proteins were pulled down by GST-TRIP6-LTWT. From the results, which are presented in Fig. 6C, we can conclude that the third PDZ domain of Scrib is required for an efficient interaction with TRIP6, since binding to GST-TRIP6-LTWT was almost completely abolished when the carboxylate binding loop of this PDZ domain was destroyed.
Fig. 4. Scrib interacts with TRIP6 in vivo but not with zyxin, ajuba or LIMD1. CV-1 cells were transiently cotransfected with Xpress-hScribmito or Xpress-hScribdPDZ-mito, and GFP-fusions of full length human TRIP6, zyxin, ajuba, or LIMD1 as indicated. Cells were stained with an anti-Xpress antibody to detect Xpress-hScrib-mito (left panels). GFP fluorescence was visualized by epifluorescence microscopy (right panels). A focal plane corresponding to mitochondrial staining is shown.
To find out which of the four PDZ domains of Scrib is required for binding to TRIP6, we performed additional GST pull-down experiments. We mutated the PDZ domains of Scrib, one at the time, by destroying their carboxylate
3.4. Scrib is dispensable for targeting TRIP6 to cell–cell contacts and to focal adhesions Scrib has been reported to be localized in cell–cell contacts [19], and also for TRIP6, it was shown that it is localized in these structures [33]. Previous studies have shown that PDZ proteins play an important role in the targeting of proteins to specific membrane compartments. From our studies [12], we know that the Scrib protein is expressed in MDCKII cells. Therefore, we investigated whether Scrib was essential to localize TRIP6 in cell–cell contacts in MDCKII cells. For this, we made stable MDCKII cell lines expressing wild-type and mutated GFP-coupled forms of the TRIP6 protein, of which the mutant form is not able to bind anymore to Scrib. If Scrib would be essential to target TRIP6 to cell–cell contacts, then wild-type GFP-TRIP6 would be recruited to cell–cell contacts by the endogenously present Scrib while the mutated GFPTRIP6 form, which is not able to bind to Scrib, would not be recruited to these structures anymore. However, as shown in Fig. 7 (upper panels), mutated GFP-TRIP6 proteins were still able to localize in cell–cell contacts in a similar way as their wild-type GFP-TRIP6 counterparts. These results suggest that Scrib is not responsible for targeting TRIP6 to cell–cell contacts. Since TRIP6 is also localized in focal adhesions, we also investigated whether Scrib was necessary to target TRIP6 to these structures. From our studies [12], we know that the Scrib protein is expressed in CV-1 cells. Therefore, we investigated whether Scrib was essential to localize TRIP6 in focal adhesions in CV1 cells. For this, CV-1 cells were transiently transfected with a construct expressing GFP-TRIP6WT containing full length wild-type TRIP6, or GFP-TRIP6T474A, which is identical to GFP-TRIP6WT except for a point mutation replacing threonine474 by alanine, which abolishes binding to Scrib. If Scrib would be essential to target TRIP6 to focal adhesions, then wild-type GFP-TRIP6 would be targeted to these structures by the endogenously present Scrib proteins, while the GFPTRIP6T474A mutated form, which is not able to bind to Scrib anymore, would not be targeted to focal adhesions. However, as shown in Fig. 7 (lower panels), no difference in focal adhesion localization could be detected between wild-type and mutated
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Fig. 7. Scrib is dispensable for targeting TRIP6 to focal adhesions and to cell–cell contacts. (upper panels) CV-1 cells were transiently transfected with GFP-coupled human TRIP6 proteins as indicated. GFP fusions were visualized by epifluorescence microscopy. (lower panels) MDCKII cells stably expressing GFP-coupled human TRIP6 proteins as indicated were analyzed for GFP-fluorescence by confocal microscopy.
Fig. 6. TRIP6 directly interacts with Scrib in vitro. (A) GST-fusion proteins, as indicated in the figure, and GST alone were expressed in E. coli, purified and analyzed by SDS–PAGE and Coomassie Blue staining. LT: LIM domains and C-terminal tail. (B) In vitro synthesized [35S] methionine-labeled Scrib was incubated with immobilized GST or with GST fusion proteins as indicated. After extensive washing, bound proteins were separated by SDS–PAGE and visualized by autoradiography. The amount of synthesized protein loaded as a reference on the gel corresponds to 10% of the input used in each binding experiment. (C) All four PDZ domains of Scrib (amino acids 616–1490), either wild-type or mutated as indicated, were synthesized in vitro and [35S] methionine-labeled. These labeled proteins were incubated with immobilized GST or with GST-TRIP6-LTWT and allowed to interact over night at 4 C. After extensive washing, bound proteins were eluted in sample buffer, separated by SDS–PAGE and visualized by autoradiography. The amount of synthesized protein loaded as a reference on the gel corresponds to 1% of the input used in each binding experiment.
GFP-TRIP6 proteins. These results indicate that Scrib is dispensable for targeting TRIP6 to focal adhesions.
4. Discussion In a previous study, we found that one of zyxinÕs family members, LPP, interacts with the tumor suppressor Scrib. In this study, we report that, of the four remaining zyxin family members, only TRIP6 also specifically interacts with Scrib, and that Scrib does not bind to zyxin, ajuba and LIMD1.
We provide evidence that the interaction between TRIP6 and Scrib is mediated by the carboxy-terminus of TRIP6 and the third PDZ domain of Scrib. In the course of our studies, we also have functionally characterized the nuclear export signal in the human TRIP6 protein, and found that LIMD1 localizes in focal adhesions. Most information regarding the function of Scrib comes from studies in Drosophila melanogaster. Drosophila Scribble was identified as being required for the apical confinement of polarity determinants in epithelia [20]. Mutations in Scribble cause aberrant cell shapes and loss of the monolayer organization in embryonic epithelia. Loss of Scribble function results in the misdistribution of apical proteins and adherens junctions to the basolateral cell surface. Subsequent studies in Drosophila provided evidence that Scribble is a tumor suppressor and cooperates with two other tumor suppressors, Lethal giant larvae (Lgl) and Discs-large (Dlg) to regulate cell polarity and growth control [21]. A recent study of the Drosophila genome sequence revealed that zyxin, LPP and TRIP6 form a paralogous group in vertebrates that is represented by the zyx102 protein in D. melanogaster [11]. Examination of the carboxy-terminal tail (-TSEH) of the zyx102 protein reveals that it contains a serine at the 2 position and a histidine at the 0 position. Therefore, it is likely that the zyx102 protein interacts with Scribble, and that this feature of Scribble/Zyx102-binding was retained in evolution by LPP and TRIP6 but was lost by zyxin. In this regard, it is interesting to note that the CG11063 protein, which represents ajuba and LIMD1 in Drosophila, has a completely different carboxy-terminus (-QYMG) that most likely does not bind to Scribble. If this would be indeed the case, the situation in Drosophila would be similar to the one in mammalian cells where we have shown that Scrib does not bind to ajuba and LIMD1. Follow-up studies are necessary to investigate this hypothesis and the hypothesis that Scribble binds to Zyx102. If Scribble indeed binds to Zyx102 then it can be expected that the Drosophila ancestor of zyxin, LPP and TRIP6 is involved in one way or another in the above-described Scribble-associated functions.
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It is not clear, what are the functional implications of the interaction between Scrib and LPP/TRIP6. Our finding that Scrib selectively binds to LPP and TRIP6 but not to any of the other zyxin family members, suggests that LPP and TRIP6 perform a function in cells, which is not carried out by the other family members. Of all zyxinÕs family members, TRIP6 is the one that has the highest degree of similarity to LPP, based on sequence comparison. The LIM domains of TRIP6 show 62%, 77% and 79% amino acid identity to the ones of LPP, and in the proline-rich pre-LIM region, LPP and TRIP6 have two regions in common with high sequence similarity, which are not present in any of the other family members. The function of these regions remains to be established but preliminary evidence from our laboratory suggests that one of these regions is a protein interaction domain. Previous studies have demonstrated that PDZ domain proteins play an important role in the targeting of proteins to specific subcellular compartments [25]. As shown before, TRIP6 is localized in focal adhesions and in cell–cell contacts [30,33]. Therefore, we investigated whether Scrib was essential to localize TRIP6 in these structures. However, this appeared not to be the case. These results are similar to what has been reported for LPP [12]. Furthermore, as previously demonstrated [12], the PDZ domains of Scrib are dispensable for its targeting to cell–cell contacts, implicating that LPP nor TRIP6 play a role in targeting Scrib to these structures. We have to mention here that preliminairy experiments from our laboratory (our unpublished results of mammalian two-hybrid experiments) suggest that the TRIP6 protein can form homodimers, and also can interact with the LPP protein, which is endogenously present in CV-1 cells [31] as well as in MDCKII cells [12], the two cell lines that were used for the targeting experiments presented here. At first sight, these findings may complicate the interpretation of these targeting experiments. Indeed, we conclude that Scrib is dispensable for targeting TRIP6 to cell–cell contacts and focal adhesions, given the fact that mutant TRIP6 molecules, which are not able to directly bind to Scrib anymore, are still recruited to these structures. However, because of the possible homo/hetero-dimerization of the TRIP6 protein, these mutant TRIP6 molecules might still be indirectly recruited to focal adhesions and cell–cell contacts by Scrib by binding to their wild-type counterparts, i.e., endogenously present wild-type TRIP6 and/or LPP molecules. However, the mitochondrial targeting experiments that are presented here, argue against this possibility. If mutated TRIP6 proteins, which are unable to directly bind to Scrib, would be able to be recruited by Scrib by binding to their endogenously present wild-type counterparts, these mutants would likely also be recruited by Scrib in the mitochondrial targeting experiments. Results of these latter experiments, however, suggest that this is not the case, since mutant TRIP6 molecules were not recruited by Scrib on the mitochondria. TRIP6 has been reported to bind to two other PDZ domain containing proteins: the adaptor protein RIL and the protein tyrosine phosphatase mPTP-BL/hPTP1E [33,34]. Concerning Scrib, to date, two other proteins have been described that bind to the PDZ domains of Scrib: the high-risk human papillomavirus E6 protein [17] interacts with the PDZ domains of human Scrib, whereas the GUKH (guanylate kinase holder) protein was shown to bind to the PDZ domains of Scribble at Drosophila synapses [35]. These findings raise the possibility that different binding partners of the Scrib PDZ domains,
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including LPP and TRIP6, can compete with each other for binding to Scrib, and as such play a role in processes in which Scrib is involved. As previously described, the binding of LPP to Scrib appears to be regulated: in mitochondrial targeting experiments it was noticed that in the majority of the cells, full length LPP was not recruited by Scrib while a truncated version of the LPP protein, containing a deletion of the pre-LIM region was efficiently recruited [12]. It was hypothesized that the binding of LPP to Scrib is regulated by an intra- or intermolecular interaction of LPP, as a result of which the carboxy-terminal tail is hidden in such a way that it is not available anymore for binding to Scrib. These results are in sharp contrast to the results obtained with full length TRIP6: full length TRIP6 was recruited to Scrib-coated mitochondria in nearly 100% of the cells examined. These results may suggest that although both TRIP6 and LPP mainly bind to the third PDZ domain of Scrib ([12] and results presented here), their interaction with Scrib might serve a different function. The demonstration that two of zyxinÕs family members, which shuttle between the nucleus and the cytoplasm, are present in the same molecular complex as Scrib, links Scrib to a communication pathway between cell–cell contacts and the nucleus, and implicates zyxin family members in Scribassociated functions. Acknowledgements: We thank Sandra Meulemans for excellent technical assistance, and Jan Brants, Christopher Huggins, Hajnalka Kiss, and Tyson Sharp for interesting discussions. This work was supported in part by GOA (Geconcerteerde Onderzoeksacties) Grant 2002/10, and by grants from the Cancer Research Program of ‘‘Fortis Verzekeringen’’, the Fund for Scientific Research (F.W.O.-Vlaanderen Krediet Aan Navorsers, 1.5.098.03), the Belgian Federation against Cancer (project A5890), and the University VIS program (project 99/010). Marleen Petit is a Postdoctoral Fellow of the Fund for Scientific Research – Flanders (Belgium)(F.W.O.-Vlaanderen).
References [1] Petit, M.M.R., Fradelizi, J., Golsteyn, R.M., Ayoubi, T.A.Y., Menichi, B., Louvard, D., Van de Ven, W.J.M. and Friederich, E. (2000) LPP, an actin cytoskeleton protein related to zyxin, harbors a nuclear export signal and transcriptional activation capacity. Mol. Biol. Cell. 11, 117–129. [2] Nix, D.A. and Beckerle, M.C. (1997) Nuclear-cytoplasmic shuttling of the focal contact protein, zyxin: a potential mechanism for communication between sites of cell adhesion and the nucleus. J. Cell Biol. 138, 1139–1147. [3] Wang, Y. and Gilmore, T.D. (2001) LIM domain protein Trip6 has a conserved nuclear export signal, nuclear targeting sequences, and multiple transactivation domains. Biochim. Biophys. Acta 1538, 260–272. [4] Kanungo, J., Pratt, S.J., Marie, H. and Longmore, G.D. (2000) Ajuba, a cytosolic LIM protein, shuttles into the nucleus and affects embryonal cell proliferation and fate decisions. Mol. Biol. Cell 11, 3299–3313. [5] Huggins, C.J., Gokgoz, N., Dimaras, H. and Andrulis, I.L. (2003) Molecular and genetic analysis of the LIMD1 gene. In: Proceedings of the 94th Annual Meeting of the American Association for Cancer Research, vol. 44, p. 532. [6] Beckerle, M.C. (1986) Identification of a new protein localized at sites of cell-substrate adhesion. J. Cell Biol. 103, 1679–1687. [7] Yi, J. and Beckerle, M.C. (1998) The human TRIP6 gene encodes a LIM domain protein and maps to chromosome 7q22, a region associated with tumorigenesis. Genomics 49, 314–316. [8] Petit, M.M.R., Mols, R., Schoenmakers, E.F., Mandahl, N. and Van de Ven, W.J.M. (1996) LPP, the preferred fusion partner
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M.M.R. Petit et al. / FEBS Letters 579 (2005) 5061–5068 gene of HMGIC in lipomas, is a novel member of the LIM protein gene family. Genomics 36, 118–129. Goyal, R.K., Lin, P., Kanungo, J., Payne, A.S., Muslin, A.J. and Longmore, G.D. (1999) Ajuba, a novel LIM protein, interacts with Grb2, augments mitogen-activated protein kinase activity in fibroblasts, and promotes meiotic maturation of Xenopus oocytes in a Grb2- and Ras-dependent manner. Mol. Cell Biol. 19, 4379– 4389. Kiss, H. et al. (1999) A novel gene containingLIM domains (LIMD1) is located within the common eliminated region 1 (C3CER1) in 3p21.3. Hum. Genet. 105, 552–559. Renfranz, P.J., Siegrist, S.E., Stronach, B.E., Macalma, T. and Beckerle, M.C. (2003) Molecular and phylogenetic characterizationof Zyx102, a Drosophila orthologue of the zyxin family that interacts with Drosophila Enabled. Gene 305, 13–26. Petit, M.M.R., Meulemans, S.M.P., Alen, P., Ayoubi, T.A.Y., Jansen, E. and Van de Ven, W.J.M. (2005) The tumor suppressor scrib interacts with the zyxin-related protein LPP, which shuttles between cell adhesion sites and the nucleus. BMC Cell Biol. 6, 1. Bilder, D. et al. (2000) Collective nomenclature for LAP proteins. Nat. Cell Biol. 2, E114. Apperson, M.L., Moon, I.S. and Kennedy, M.B. (1996) Characterization of densin-180, a new brainspecific synaptic protein of the O-sialoglycoprotein family. J. Neurosci. 16, 6839–6852. Borg, J.P. et al. (2000) ERBIN: a basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor. Nat. Cell Biol. 2, 407–414. Saito, H. et al. (2001) Lano, a novel LAP protein directly connected to MAGUK proteins in epithelial cells. J. Biol. Chem. 5, 5. Nakagawa, S. and Huibregtse, J.M. (2000) Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitinprotein ligase. Mol. Cell. Biol. 20, 8244–8253. Santoni, M.J., Pontarotti, P., Birnbaum, D. and Borg, J.P. (2002) The LAP family: a phylogenetic point of view. Trends Genet. 18, 494–497. Dow, L.E. et al. (2003) hScrib is a functional homologue of the Drosophila tumour suppressor Scribble. Oncogene 22, 9225–9230. Bilder, D. and Perrimon, N. (2000) Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. Nature 403, 676–680. Bilder, D., Li, M. and Perrimon, N. (2000) Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113–116. Nguyen, M.M., Nguyen, M.L., Caruana, G., Bernstein, A., Lambert, P.F. and Griep, A.E. (2003) Requirement of PDZ-
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containing proteins for cell cycle regulation and differentiation in the mouse lens epithelium. Mol. Cell. Biol. 23, 8970–8981. Sadowski, I., Bell, B., Broad, P. and Hollis, M. (1992) GAL4 fusion vectors for expression in yeast or mammalian cells. Gene 118, 137–141. Buchert, M., Schneider, S., Adams, M.T., Hefti, H.P., Moelling, K. and Hovens, C.M. (1997) Useful vectors for the two-hybrid system in mammalian cells. Biotechniques 23, 396–398, 400, 402. Hung, A.Y. and Sheng, M. (2002) PDZ domains: structural modules for protein complex assembly. J. Biol. Chem. 277, 5699– 5702. Songyang, Z. et al. (1997) Recognition of unique carboxylterminal motifs by distinct PDZ domains. Science 275, 73–77. Yi, J., Kloeker, S., Jensen, C.C., Bockholt, S., Honda, H., Hirai, H. and Beckerle, M.C. (2002) Members of the zyxin family of LIM proteins interact with members of the p130Cas family of signal transducers. J. Biol. Chem. 277, 9580–9589. Pistor, S., Chakraborty, T., Niebuhr, K., Domann, E. and Wehland, J. (1994) The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton. EMBO J. 13, 758–763. Bubeck, P., Pistor, S., Wehland, J. and Jockusch, B.M. (1997) Ligand recruitment by vinculin domains in transfected cells. J. Cell Sci. 110, 1361–1371. Wang, Y., Dooher, J.E., Zhao, M. and Gilmore, T.D. (1999) Koedood Characterization of mouse Trip6: a putative intracellular signaling protein. Gene 234, 403–409. Petit, M.M.R., Meulemans, S.M.P. and Van De Ven, W.J.M. (2003) The focal adhesion and nuclear targeting capacity of the LIM-containing Lipoma-preferred partner (LPP) protein. J. Biol. Chem. 278, 2157–2168. Marie, H. et al. (2003) The LIM protein Ajuba is recruited to cadherin-dependent cell junctions through an association with alpha-catenin. J. Biol. Chem. 278, 1220–1228. Cuppen, E., van Ham, M., Wansink, D.G., de Leeuw, A., Wieringa, B. and Hendriks, W. (2000) The zyxin-related protein TRIP6 interacts with PDZ motifs in the adaptor protein RIL and the protein tyrosine phosphatase PTP-BL. Eur. J. Cell Biol. 79, 283–293. Murthy, K.K., Clark, K., Fortin, Y., Shen, S.H. and Banville, D. (1999) ZRP-1, a zyxin-related protein, interacts with the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E. J. Biol. Chem. 274, 20679–20687. Mathew, D., Gramates, L.S., Packard, M., Thomas, U., Bilder, D., Perrimon, N., Gorczyca, M. and Budnik, V. (2002) Recruitment of scribble to the synaptic scaffolding complex requires GUK-holder, a novel DLG binding protein. Curr. Biol. 12, 531–539.
FEBS Letters 579 (2005) 5061–5068
The tumor suppressor Scrib selectively interacts with specific members of the zyxin family of proteins Marleen M.R. Petit, Koen R.M.O. Crombez, Hilke B.V.K. Vervenne, Nancy Weyns, Wim J.M. Van de Ven* Laboratory for Molecular Oncology, Department of Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology (VIB), Herestraat 49, B-3000 Leuven, Belgium Received 12 January 2005; revised 7 July 2005; accepted 9 August 2005 Available online 19 August 2005 Edited by Berend Wieringa
Abstract The zyxin family of proteins consists of five members, ajuba, LIMD1, LPP, TRIP6 and zyxin, which localize at cell adhesion sites and shuttle to the nucleus. Previously, we established that LPP interacts with the tumor suppressor Scrib, a member of the leucine-rich repeat and PDZ (LAP) family of proteins. Here, we demonstrate that Scrib also interacts with TRIP6, but not with zyxin, ajuba, or LIMD1. We show that TRIP6 directly binds to the third PDZ domain of Scrib via its carboxy-terminus. Both proteins localize in cell–cell contacts but are not responsible to target each other to these structures. In the course of our experiments, we also characterized the nuclear export signal of human TRIP6, and show that LIMD1 is localized in focal adhesions. The binding between two of zyxinÕs family members and Scrib links Scrib to a communication pathway between cell–cell contacts and the nucleus, and implicates these zyxin family members in Scrib-associated functions. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Lano; Erbin; Densin-180; Let-413; Scribble; KIAA0147
1. Introduction At sites of cell adhesion, an increasing number of proteins not only performs structural tasks, such as sustaining cell shape, but is also involved in signaling events. A family of proteins that may operate as such is the zyxin family of LIM domain proteins. Members of this family localize at sites of cell adhesion, such as focal contacts, which are membrane attachment sites to the extracellular matrix, and cell–cell contacts. However, apart from this localization in cell adhesion sites, these proteins also transiently localize in the nucleus [1–5]. The zyxin family consists of five members: zyxin [6], TRIP6 (thyroid receptor interacting protein 6) [7], LPP (lipoma pre* Corresponding author. Fax: +32 16 34 60 73. E-mail address: [email protected] (W.J.M. Van de Ven).
Abbreviations: GFP, green fluorescent protein; GST, glutathione S-transferase; LAP, leucine-rich repeat and PDZ; LIMD1, LIM domain containing 1; LPP, lipoma preferred partner; LRR, leucine-rich repeat; NES, nuclear export signal; PBS, phosphate buffered saline; PDZ, PSD95/discs-large/ZO-1; TRIP6, thyroid receptor interacting protein 6
ferred partner) [8], ajuba [9] and LIMD1 (LIM domain containing 1) [10]. A recent study of the Drosophila genome sequence indicates that zyxin, LPP, and TRIP6 form one paralogous group in vertebrates that is represented by the zyx102 protein in the fruit fly, while ajuba and LIMD1 form another group that is represented by the CG11063 protein (Fig. 1) [11]. All family members share a similar organization having three LIM domains (zinc-finger protein interaction domains) at their carboxy-terminus, which is preceded by a proline-rich preLIM region containing a number of protein interaction domains. While all family members have a high degree of amino acid similarity in their LIM domains, their pre-LIM regions are quite divergent. Because of their structural features and their characteristic to shuttle between the nucleus and the cytoplasm, zyxin family members have been proposed to be scaffolding proteins involved in signal transduction from sites of cell adhesion to the nucleus. In a previous study, we identified the tumor suppressor Scrib as an interaction partner of one of zyxinÕs family members, LPP [12]. Scrib is a member of the LAP (LRR (leucine-rich repeat) and PDZ (PSD-95/discs-large/ZO-1)) family of membrane-associated proteins [13], which consists of four members: Densin-180, Erbin, Lano, and Scrib [14–17]. LAP proteins contain a set of LRRs at their amino-terminus, and four (Scrib), one (Erbin, Densin-180) or no (Lano) copies of the PDZ domain. A specific characteristic of these proteins are the LAP-specific domains (LAPSa and b), which are located carboxy-terminally of the LRRs [18]. LPP binds to the PDZ domains of Scrib via its C-terminus. Little information is available at the moment about the function of Scrib. Relating to the control of cell polarity and proliferation, Scrib is a functional homologue of the Drosophila scribble protein [19]. Polarity defects [20] and tumorous overgrowth in Scribblemutant flies [21] are rescued by Scrib predicting an important role for Scrib in the suppression of mammalian tumorigenesis. This hypothesis is further supported by the finding that highrisk papilloma virus E6 proteins direct Scrib for degradation by directly binding to the PDZ-domains of Scrib [17,22]. Here, we report about the differential interaction between Scrib and members of the zyxin family. We found that Scrib, besides with LPP, also selectively interacts with TRIP6, but not with zyxin, ajuba or LIMD1. The binding between Scrib and TRIP6 is mediated by the third PDZ domain of Scrib and the C-terminus of TRIP6. Furthermore, we have studied whether Scrib is involved in the subcellular targeting of TRIP6 to focal adhesions and to cell–cell contacts.
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.08.012
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M.M.R. Petit et al. / FEBS Letters 579 (2005) 5061–5068 containing all four PDZ domains) (wild-type or mutated) in NENT100 buffer (100 mM NaCl, 20 mM Tris–HCl, pH 7.6, 1 mM EDTA, 0.1% NP-40, protease inhibitors). This mixture was tumbled overnight at 4 C. Subsequently, the beads were washed 5 times in 500 ll NENT100 buffer, resuspended in 25 ll SDS–PAGE sample buffer. Proteins were separated by SDS–PAGE and interacting Scrib was detected by autoradiography.
Fig. 1. Schematic phylogenetic representation of zyxin family members with their C-termini and requirements for binding to Scrib-type PDZ domains. h, hydrophobic amino acid.
2. Materials and methods 2.1. Plasmid constructs Constructs expressing wild-type or mutated human zyxin, TRIP6, ajuba, and LIMD1 as green fluorescent protein (GFP) fusions were made by cloning the coding regions in the pEGFP-C vectors (Clontech). TRIP6 en zyxin cDNAs, and ajuba and LIMD1 cDNAs were kindly provided by Mary Beckerle (University of Utah, USA) and Greg Longmore (Washington University School of Medicine, USA), respectively. The constructs expressing Xpress-hScrib-mito and Xpress-hScribdPDZ-mito were described before [12]. Mutations were made using the QuikChangee site-directed Mutagenesis Kit (Stratagene). All synthetic mutations, ligation sites and PCR-amplified regions were verified by sequencing. 2.2. Cell culture, stable cell lines and transfections Cell lines included CV-1 (ATCC CCL-70), HEK293 (ATCC CRL1573), 293T (HEK 293 cells expressing SV40 T-antigen), and MDCK strain II (Dog normal kidney epithelial cells). Cells were grown in DMEM/F12 (1:1) (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and cultured at 37 C in a humidified CO2 incubator. Transient transfections were performed using FuGenee 6 Transfection Reagent (Roche). Stable MDCKII cell lines were made expressing wild-type and carboxy-terminally mutated human GFP-TRIP6 proteins. Transfection of MDCK cells was performed using Lipofectamine 2000 Reagent (Life Technologies). Transfected cells were selected in medium containing 250 lg/ml G418 (Life Technologies), and resistant colonies were isolated 10–14 days later. Individual clones were screened for expression of the GFP fusion proteins by Western blotting using a rabbit polyclonal anti-GFP antibody (Tebu Bio). 2.3. Mammalian two-hybrid system Bait- and prey-constructs were made using pM-vectors [23] and the pSNATCH-vector [24], respectively. 24 h upon seeding, semi-confluent HEK293 cells on 24-well plates were transiently cotransfected with 100 ng bait-DNA, 100 ng prey-DNA, 250 ng DNA of a luciferase reporter construct [12] and 50 ng of CMV-b-galactosidase DNA (internal control for transfection efficiency). Cell lysates were prepared 18–24 h after transfection and assayed for luciferase activity as described previously [1]. 2.4. In vitro transcription/translation and GST pull-down assays All in vitro translation reactions were carried out using the TNT T7 Quick Coupled Transcription/Translation System (Promega). For GST pull-down assays, bacterial expression constructs were made using pGEX-5X vectors (Amersham-Pharmacia Biotech) directing the synthesis of glutathione S-transferase (GST) fusion proteins containing wild-type or mutated forms of human TRIP6. These fusion proteins were purified according to manufacturerÕs instructions and verified by SDS–PAGE. GST fusion proteins or GST alone, bound to glutathione–agarose beads, were incubated with in vitro synthesized [35S] methionine-labeled human Scrib protein (amino acids 616–1490
2.5. GFP-fluorescence and indirect immunocytochemistry CV-1 or 293T cells seeded on glass coverslips, and MDCKII cells seeded on Transwell-Clear polyester membranes (0.4 lm, Costar) were fixed in 4% formaldehyde for 20 min at room temperature followed by three washes in phosphate buffered saline (PBS) containing 0.1 mM CaCl2 and 0.1 mM MgCl2 (PBS++). For GFP-fluorescence, slides were mounted in vectashield mounting medium (Vector Laboratories, Inc.) and analyzed on a Zeiss Axiophot fluorescence microscope equipped with an RT slider SPOT camera (Diagnostic Instruments, Inc.) using SPOT RT Software v3.4, or by confocal microscopy (MRC-1024 Laser Scanning Confocal Imaging System, Bio-Rad). Indirect immunocytochemistry was done as described [12]. Primary antibodies: a rabbit polyclonal anti-GAL4 DNA-binding domain antibody (Tebu Bio), a monoclonal anti-Xpress antibody (Life Technologies), and a monoclonal anti-vinculin antibody hVIN-1 (Sigma). Secondary antibodies: fluorescently-tagged Alexa antibodies (Molecular Probes).
3. Results 3.1. Scrib binds to the C-terminus of TRIP6 via its PDZ domains In a previous study [12], we identified Scrib as an interaction partner of LPP, which is a member of the zyxin family of proteins. The binding between Scrib and LPP is mediated via the PDZ domains of Scrib and the C-terminus of LPP. The binding specificity of PDZ domains to C-termini, which is the typical mode of interaction of PDZ domains, is critically determined by the interaction of the residue at position aB1 and the side chain of the 2 residue of the C-terminal ligand [25,26]. Since all four PDZ domains of Scrib contain a histidine at position aB1, the carboxy-terminal sequence of Scrib target proteins is predicted to require a hydrophobic amino acid (h) at the 0 (carboxy-terminus) position, and a serine (S) or threonine (T) at the 2 position (Fig. 1). When comparing the carboxy-terminal tails of all human zyxin family members, we noticed that, besides LPP, also the carboxy-terminus of TRIP6 theoretically fulfils the criteria for binding to the PDZ domains of Scrib, while the C-termini of zyxin, ajuba and LIMD1 do not (Fig. 1). This means that, at least in theory, Scrib would be able to bind to the C-terminus of TRIP6 but not to the C-terminus of zyxin, ajuba or LIMD1. To evaluate these predictions experimentally, we first performed a mammalian twohybrid experiment. We used as baits a C-terminal portion of all zyxin family members, consisting of the LIM-domains and the C-terminal tail of the respective proteins. These baits were designated as follows: pM-TRIP6LT, pM-zyxinLT, pMajubaLT and pM-LIMD1LT. As prey-protein, we used pSNATCH-hScribPDZ containing a part of the human Scrib protein (amino acids 669–1233) surrounding all four PDZ-domains. As summarized in Fig. 2A, the interaction between TRIP6 and Scrib resulted in high levels of luciferase reporter activity. These high levels were not obtained when using the baits containing zyxin, ajuba, or LIMD1. These results suggest that, besides with LPP, Scrib also interacts with TRIP6 but not with zyxin, ajuba or LIMD1.
M.M.R. Petit et al. / FEBS Letters 579 (2005) 5061–5068
Fig. 2. Scrib binds to the C-terminus of TRIP6 via its PDZ domains. pM-bait- and pSNATCH-prey-constructs were cotransfected into 293 cells in the combination indicated (LT: LIM domains and carboxyterminal tail), together with a GAL4-regulated luciferase reporter and a CMV-b-galactosidase internal control. Cell lysates were assayed for luciferase activity 18–24 h after transfection. Relative luciferase activity is reported as the average of three independent duplo experiments (with standard error).
Interaction between bait- and prey-proteins in a mammalian two-hybrid assay takes place in the nucleus. For an accurate performance of this assay, this means that bait- and preyproteins should be accumulating in the nucleus. In the abovedescribed mammalian two-hybrid experiment, where we used partial bait proteins, all baits, also the ones of zyxin, ajuba and LIMD1, were well expressed on Western blot and were accumulating in the nucleus of the cells (results not shown). In a follow-up experiment, we wanted to confirm the interaction between TRIP6 and Scrib, and to determine the amino acids in TRIP6 that are critical for its interaction with the PDZ domains of TRIP6, however, this time using the full length TRIP6 protein as a bait. Since TRIP6 contains a nuclear export signal (NES) in its pre-LIM region, we had to use bait-proteins in which this NES had been inactivated. However, for TRIP6, a NES is only characterized for the mouse protein [3]. As it was reported for zyxin that it has one or two NESs depending on the species [27], we wanted to evaluate first whether the NES that had been mapped in the mouse TRIP6 protein was also the NES of the human protein. In mouse TRIP6, three leucine residues are critical for nuclear export (Fig. 3A). When these residues are mutated into alanine residues, nuclear export of mouse TRIP6 is abolished
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[3]. We mutated the corresponding leucine residues in the human TRIP6 protein into alanine residues in a GFP-hTRIP6WT fusion (Fig. 3A). Wild-type and mutated proteins were introduced into CV-1 fibroblast cells. While GFP-TRIP6WT, containing full length wild-type human TRIP6 with an intact NES, was excluded from the nucleus (Fig. 3B, left panel), GFP-TRIP6dNES, containing full length human TRIP6 with the above-described leucine-to-alanine mutations, was accumulating in the nucleus (Fig. 3B, right panel). To verify whether mutation of the NES in human TRIP6 also induced nuclear accumulation of the bait-protein that was used in the mammalian two-hybrid assay, we introduced wild-type and mutated TRIP6-bait-proteins in 293T cells. While pMTRIP6WT, containing GAL4-fused full length wild-type human TRIP6 with an intact NES, was excluded from nuclei (Fig. 3C, upper panels), pM-TRIP6dNES, containing GAL4fused full length human TRIP6 with the above-described leucine-to-alanine mutations, was accumulating in the nuclei of the cells (Fig. 3C, lower panels). These results indicate that, not only in mouse but also in human TRIP6, the leucines at positions 100, 104 and 107 are critical for proper nuclear export of the protein. To perform the follow-up two-hybrid experiment, we used as baits for TRIP6, pM-TRIP6dNES, and pM-TRIP6dNES,T474A and pM-TRIP6dNES,C476A, which are identical to pMTRIP6dNES except for a point mutation to alanine introduced at threonine474 (position 2) and cysteine476 (position 0), respectively, the two residues that are theoretically critical for interaction with the Scrib PDZ domains. pSNATCHhScribPDZ was again used as prey. As summarized in Fig. 2B, the interaction between wild-type full length TRIP6 and the Scrib PDZ domains resulted in high levels of luciferase reporter activity. These high levels dropped to background when pM-TRIP6dNES,T474A or pM-TRIP6dNES,C476A were used as baits. The ‘‘background’’ levels of luciferase that were detected when pM-TRIP6-baits were used in combination with pSNATCH (empty prey-vector) as prey, are due to the intrinsic transcriptional activation activity of the human TRIP6 protein, as has been shown for mouse TRIP6 [3]. These results indicate that TRIP6 binds to the PDZ domains of Scrib and that amino acids at position 0 and 2 are critical for this interaction. 3.2. Scrib interacts with LPP and TRIP6 in vivo but not with zyxin, ajuba or LIMD1 Evidence for an in vivo interaction between Scrib and TRIP6 was obtained by performing mitochondrial targeting experiments. We tested if Scrib was sufficient to recruit TRIP6 to an ectopic location in vivo. The membrane anchor of the ActA sequence has been shown previously to be sufficient to target proteins expressed in mammalian cells to the surface of mitochondria [28,29]. This ectopic localization allows testing ligand recruitment in vivo. For this purpose, we generated a chimera named Xpress-hScrib-mito made up by an Xpress-epitope tag fused to the amino-terminus of human full length Scrib and linked in frame to the membrane anchor of the Listeria monocytogenes protein ActA (mito). CV-1 cells were transiently transfected with Xpress-hScribmito and full length wild-type or carboxy-terminally mutated TRIP6 GFP-fusions. Cells were labeled with an anti-Xpress antibody and examined by fluorescence microscopy. As shown in Fig. 4A, the Xpress-hScrib-mito chimera localized
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Fig. 3. Mapping of the NES in human TRIP6. (A) Alignment of a portion of the LPP amino acid sequence with the corresponding sequence in mouse and human TRIP6. The boxed region in LPP and the boxed leucine residues in mouse TRIP6 were previously shown to be essential for nuclear export of the respective proteins [1,3]. The boxed leucine residues in human TRIP6 were mutated into alanines in the experiments presented here, and shown to be part of a unique NES in the human TRIP6 protein. (B) CV-1 fibroblasts were transiently transfected with GFP-hTRIP6 proteins as indicated. GFP fusions were visualized by epifluorescence microscopy. A focal plane corresponding to the cell body is shown. Insets are focused on focal adhesions. (C) 293T epithelial cells were transiently transfected with pM-hTRIP6 proteins as indicated. pM-hTRIP6 fusions were stained with anti-GAL4 antibodies and visualized by epifluorescence microscopy. Nuclei were visualized by staining with DAPI.
to mitochondria. While wild-type TRIP6 was recruited to Xpress-hScrib-mito on mitochondria, this recruitment was abolished when the carboxy-terminus of TRIP6 was mutated (Fig. 4A). To investigate the importance of the PDZ domains of Scrib in this recruitment of TRIP6, we deleted all four PDZ domains (amino acids 724-1192) from Xpress-hScrib-mito (= Xpress-hScribdPDZ-mito), and tested whether this PDZless protein still was able to recruit TRIP6 to mitochondria. As shown in Fig. 4A, lower panels, Xpress-hScribdPDZ-mito lost its ability to recruit TRIP6 to mitochondria. These results indicate that the PDZ domains of Scrib are an absolute requirement for its binding to TRIP6. We next investigated whether the remaining family members of LPP and TRIP6 could be recruited to Xpress-Scrib-mitocoated mitochondria. CV-1 cells were cotransfected with Xpress-hScrib-mito and full length human zyxin, ajuba, and LIMD1 GFP-fusions respectively. As shown in Fig. 4B, no recruitment could be detected. Interesting to note here is that we could easily detect GFP-LIMD1 in, what looked like, focal adhesions. To find out whether GFP-LIMD1 indeed localizes to focal adhesions, we performed double labeling experiments. CV-1 cells were transfected with GFP-LIMD1, and labeled with vinculin antibodies used as a marker for focal adhesions. As shown in Fig. 5, GFP-LIMD1 indeed colocalized with vin-
culin in focal adhesions. To our knowledge, this is a novel finding, and taken together with what has been published for zyxin [6], TRIP6 [30], LPP [1], and ajuba [31,32], this indicates that all of zyxinÕs family members can localize in focal adhesions. In conclusion, our results indicate that Scrib selectively binds to two of zyxinÕs family members, being LPP and TRIP6, and not to zyxin, ajuba, and LIMD1. 3.3. Direct interaction between the carboxy-terminus of TRIP6 and the PDZ domains of Scrib To demonstrate the direct interaction between TRIP6 and Scrib-PDZ domains, we performed GST pull-down experiments. In vitro translated Scrib-PDZ domains were tested for binding with glutathione beads, which were coupled with GST-TRIP6-LTWT, GST-TRIP6-LTC476A, or GST alone. GST-TRIP6-LTWT contains the three LIM domains and the wild-type carboxy-terminal tail of human TRIP6. GSTTRIP6-LTC476A is identical to GST-TRIP6-LTWT except for a point mutation to alanine introduced at cysteine476 (position 0). All GST-fusion proteins as well as GST alone were expressed well in Escherichia coli (Fig. 6A). As shown in Fig. 6B, ScribPDZ domains interacted specifically with the wild-type TRIP6 protein but not with its mutated form, GST-TRIP6-LTC476A, or with GST alone. These results indicate that there is a specific and direct interaction between TRIP6 and Scrib.
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Fig. 5. LIMD1 localizes in focal adhesions. CV-1 cells were transiently transfected with GFP-LIMD1, and stained with an anti-vinculin antibody to detect endogenous vinculin. GFP fluorescence (left panel) and vinculin staining (right panel) were visualized by epifluorescence microscopy.
binding loop (LG fi AE), and tested how efficiently these mutated proteins were pulled down by GST-TRIP6-LTWT. From the results, which are presented in Fig. 6C, we can conclude that the third PDZ domain of Scrib is required for an efficient interaction with TRIP6, since binding to GST-TRIP6-LTWT was almost completely abolished when the carboxylate binding loop of this PDZ domain was destroyed.
Fig. 4. Scrib interacts with TRIP6 in vivo but not with zyxin, ajuba or LIMD1. CV-1 cells were transiently cotransfected with Xpress-hScribmito or Xpress-hScribdPDZ-mito, and GFP-fusions of full length human TRIP6, zyxin, ajuba, or LIMD1 as indicated. Cells were stained with an anti-Xpress antibody to detect Xpress-hScrib-mito (left panels). GFP fluorescence was visualized by epifluorescence microscopy (right panels). A focal plane corresponding to mitochondrial staining is shown.
To find out which of the four PDZ domains of Scrib is required for binding to TRIP6, we performed additional GST pull-down experiments. We mutated the PDZ domains of Scrib, one at the time, by destroying their carboxylate
3.4. Scrib is dispensable for targeting TRIP6 to cell–cell contacts and to focal adhesions Scrib has been reported to be localized in cell–cell contacts [19], and also for TRIP6, it was shown that it is localized in these structures [33]. Previous studies have shown that PDZ proteins play an important role in the targeting of proteins to specific membrane compartments. From our studies [12], we know that the Scrib protein is expressed in MDCKII cells. Therefore, we investigated whether Scrib was essential to localize TRIP6 in cell–cell contacts in MDCKII cells. For this, we made stable MDCKII cell lines expressing wild-type and mutated GFP-coupled forms of the TRIP6 protein, of which the mutant form is not able to bind anymore to Scrib. If Scrib would be essential to target TRIP6 to cell–cell contacts, then wild-type GFP-TRIP6 would be recruited to cell–cell contacts by the endogenously present Scrib while the mutated GFPTRIP6 form, which is not able to bind to Scrib, would not be recruited to these structures anymore. However, as shown in Fig. 7 (upper panels), mutated GFP-TRIP6 proteins were still able to localize in cell–cell contacts in a similar way as their wild-type GFP-TRIP6 counterparts. These results suggest that Scrib is not responsible for targeting TRIP6 to cell–cell contacts. Since TRIP6 is also localized in focal adhesions, we also investigated whether Scrib was necessary to target TRIP6 to these structures. From our studies [12], we know that the Scrib protein is expressed in CV-1 cells. Therefore, we investigated whether Scrib was essential to localize TRIP6 in focal adhesions in CV1 cells. For this, CV-1 cells were transiently transfected with a construct expressing GFP-TRIP6WT containing full length wild-type TRIP6, or GFP-TRIP6T474A, which is identical to GFP-TRIP6WT except for a point mutation replacing threonine474 by alanine, which abolishes binding to Scrib. If Scrib would be essential to target TRIP6 to focal adhesions, then wild-type GFP-TRIP6 would be targeted to these structures by the endogenously present Scrib proteins, while the GFPTRIP6T474A mutated form, which is not able to bind to Scrib anymore, would not be targeted to focal adhesions. However, as shown in Fig. 7 (lower panels), no difference in focal adhesion localization could be detected between wild-type and mutated
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Fig. 7. Scrib is dispensable for targeting TRIP6 to focal adhesions and to cell–cell contacts. (upper panels) CV-1 cells were transiently transfected with GFP-coupled human TRIP6 proteins as indicated. GFP fusions were visualized by epifluorescence microscopy. (lower panels) MDCKII cells stably expressing GFP-coupled human TRIP6 proteins as indicated were analyzed for GFP-fluorescence by confocal microscopy.
Fig. 6. TRIP6 directly interacts with Scrib in vitro. (A) GST-fusion proteins, as indicated in the figure, and GST alone were expressed in E. coli, purified and analyzed by SDS–PAGE and Coomassie Blue staining. LT: LIM domains and C-terminal tail. (B) In vitro synthesized [35S] methionine-labeled Scrib was incubated with immobilized GST or with GST fusion proteins as indicated. After extensive washing, bound proteins were separated by SDS–PAGE and visualized by autoradiography. The amount of synthesized protein loaded as a reference on the gel corresponds to 10% of the input used in each binding experiment. (C) All four PDZ domains of Scrib (amino acids 616–1490), either wild-type or mutated as indicated, were synthesized in vitro and [35S] methionine-labeled. These labeled proteins were incubated with immobilized GST or with GST-TRIP6-LTWT and allowed to interact over night at 4 C. After extensive washing, bound proteins were eluted in sample buffer, separated by SDS–PAGE and visualized by autoradiography. The amount of synthesized protein loaded as a reference on the gel corresponds to 1% of the input used in each binding experiment.
GFP-TRIP6 proteins. These results indicate that Scrib is dispensable for targeting TRIP6 to focal adhesions.
4. Discussion In a previous study, we found that one of zyxinÕs family members, LPP, interacts with the tumor suppressor Scrib. In this study, we report that, of the four remaining zyxin family members, only TRIP6 also specifically interacts with Scrib, and that Scrib does not bind to zyxin, ajuba and LIMD1.
We provide evidence that the interaction between TRIP6 and Scrib is mediated by the carboxy-terminus of TRIP6 and the third PDZ domain of Scrib. In the course of our studies, we also have functionally characterized the nuclear export signal in the human TRIP6 protein, and found that LIMD1 localizes in focal adhesions. Most information regarding the function of Scrib comes from studies in Drosophila melanogaster. Drosophila Scribble was identified as being required for the apical confinement of polarity determinants in epithelia [20]. Mutations in Scribble cause aberrant cell shapes and loss of the monolayer organization in embryonic epithelia. Loss of Scribble function results in the misdistribution of apical proteins and adherens junctions to the basolateral cell surface. Subsequent studies in Drosophila provided evidence that Scribble is a tumor suppressor and cooperates with two other tumor suppressors, Lethal giant larvae (Lgl) and Discs-large (Dlg) to regulate cell polarity and growth control [21]. A recent study of the Drosophila genome sequence revealed that zyxin, LPP and TRIP6 form a paralogous group in vertebrates that is represented by the zyx102 protein in D. melanogaster [11]. Examination of the carboxy-terminal tail (-TSEH) of the zyx102 protein reveals that it contains a serine at the 2 position and a histidine at the 0 position. Therefore, it is likely that the zyx102 protein interacts with Scribble, and that this feature of Scribble/Zyx102-binding was retained in evolution by LPP and TRIP6 but was lost by zyxin. In this regard, it is interesting to note that the CG11063 protein, which represents ajuba and LIMD1 in Drosophila, has a completely different carboxy-terminus (-QYMG) that most likely does not bind to Scribble. If this would be indeed the case, the situation in Drosophila would be similar to the one in mammalian cells where we have shown that Scrib does not bind to ajuba and LIMD1. Follow-up studies are necessary to investigate this hypothesis and the hypothesis that Scribble binds to Zyx102. If Scribble indeed binds to Zyx102 then it can be expected that the Drosophila ancestor of zyxin, LPP and TRIP6 is involved in one way or another in the above-described Scribble-associated functions.
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It is not clear, what are the functional implications of the interaction between Scrib and LPP/TRIP6. Our finding that Scrib selectively binds to LPP and TRIP6 but not to any of the other zyxin family members, suggests that LPP and TRIP6 perform a function in cells, which is not carried out by the other family members. Of all zyxinÕs family members, TRIP6 is the one that has the highest degree of similarity to LPP, based on sequence comparison. The LIM domains of TRIP6 show 62%, 77% and 79% amino acid identity to the ones of LPP, and in the proline-rich pre-LIM region, LPP and TRIP6 have two regions in common with high sequence similarity, which are not present in any of the other family members. The function of these regions remains to be established but preliminary evidence from our laboratory suggests that one of these regions is a protein interaction domain. Previous studies have demonstrated that PDZ domain proteins play an important role in the targeting of proteins to specific subcellular compartments [25]. As shown before, TRIP6 is localized in focal adhesions and in cell–cell contacts [30,33]. Therefore, we investigated whether Scrib was essential to localize TRIP6 in these structures. However, this appeared not to be the case. These results are similar to what has been reported for LPP [12]. Furthermore, as previously demonstrated [12], the PDZ domains of Scrib are dispensable for its targeting to cell–cell contacts, implicating that LPP nor TRIP6 play a role in targeting Scrib to these structures. We have to mention here that preliminairy experiments from our laboratory (our unpublished results of mammalian two-hybrid experiments) suggest that the TRIP6 protein can form homodimers, and also can interact with the LPP protein, which is endogenously present in CV-1 cells [31] as well as in MDCKII cells [12], the two cell lines that were used for the targeting experiments presented here. At first sight, these findings may complicate the interpretation of these targeting experiments. Indeed, we conclude that Scrib is dispensable for targeting TRIP6 to cell–cell contacts and focal adhesions, given the fact that mutant TRIP6 molecules, which are not able to directly bind to Scrib anymore, are still recruited to these structures. However, because of the possible homo/hetero-dimerization of the TRIP6 protein, these mutant TRIP6 molecules might still be indirectly recruited to focal adhesions and cell–cell contacts by Scrib by binding to their wild-type counterparts, i.e., endogenously present wild-type TRIP6 and/or LPP molecules. However, the mitochondrial targeting experiments that are presented here, argue against this possibility. If mutated TRIP6 proteins, which are unable to directly bind to Scrib, would be able to be recruited by Scrib by binding to their endogenously present wild-type counterparts, these mutants would likely also be recruited by Scrib in the mitochondrial targeting experiments. Results of these latter experiments, however, suggest that this is not the case, since mutant TRIP6 molecules were not recruited by Scrib on the mitochondria. TRIP6 has been reported to bind to two other PDZ domain containing proteins: the adaptor protein RIL and the protein tyrosine phosphatase mPTP-BL/hPTP1E [33,34]. Concerning Scrib, to date, two other proteins have been described that bind to the PDZ domains of Scrib: the high-risk human papillomavirus E6 protein [17] interacts with the PDZ domains of human Scrib, whereas the GUKH (guanylate kinase holder) protein was shown to bind to the PDZ domains of Scribble at Drosophila synapses [35]. These findings raise the possibility that different binding partners of the Scrib PDZ domains,
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including LPP and TRIP6, can compete with each other for binding to Scrib, and as such play a role in processes in which Scrib is involved. As previously described, the binding of LPP to Scrib appears to be regulated: in mitochondrial targeting experiments it was noticed that in the majority of the cells, full length LPP was not recruited by Scrib while a truncated version of the LPP protein, containing a deletion of the pre-LIM region was efficiently recruited [12]. It was hypothesized that the binding of LPP to Scrib is regulated by an intra- or intermolecular interaction of LPP, as a result of which the carboxy-terminal tail is hidden in such a way that it is not available anymore for binding to Scrib. These results are in sharp contrast to the results obtained with full length TRIP6: full length TRIP6 was recruited to Scrib-coated mitochondria in nearly 100% of the cells examined. These results may suggest that although both TRIP6 and LPP mainly bind to the third PDZ domain of Scrib ([12] and results presented here), their interaction with Scrib might serve a different function. The demonstration that two of zyxinÕs family members, which shuttle between the nucleus and the cytoplasm, are present in the same molecular complex as Scrib, links Scrib to a communication pathway between cell–cell contacts and the nucleus, and implicates zyxin family members in Scribassociated functions. Acknowledgements: We thank Sandra Meulemans for excellent technical assistance, and Jan Brants, Christopher Huggins, Hajnalka Kiss, and Tyson Sharp for interesting discussions. This work was supported in part by GOA (Geconcerteerde Onderzoeksacties) Grant 2002/10, and by grants from the Cancer Research Program of ‘‘Fortis Verzekeringen’’, the Fund for Scientific Research (F.W.O.-Vlaanderen Krediet Aan Navorsers, 1.5.098.03), the Belgian Federation against Cancer (project A5890), and the University VIS program (project 99/010). Marleen Petit is a Postdoctoral Fellow of the Fund for Scientific Research – Flanders (Belgium)(F.W.O.-Vlaanderen).
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