Processing of the human protocadherin Fat1 and translocation of its cytoplasmic domain to the nucleus

Experimental Cell Research 307 (2005) 100 – 108 www.elsevier.com/locate/yexcr

Processing of the human protocadherin Fat1 and translocation of its cytoplasmic domain to the nucleus Thomas Magg, Dietmar Schreiner, Gonzalo P. Solis, Ernesto G. Bade, Hans Werner Hofer* University of Konstanz, Department of Biology, Box M648, D-78547 Konstanz, Germany Received 31 August 2004, revised version received 4 March 2005 Available online 15 April 2005

Abstract The giant protein hFat1, a member of the cadherin superfamily, has been proposed to play roles in cerebral development, glomerular slit formation, and also to act as a tumor suppressor, but its mechanisms of action have not been elucidated. To examine functions of the transmembrane and cytoplasmic domains, they were expressed in HEK293 and HeLa cells as chimeric proteins in fusion with EGFP and extracellular domains derived from E-cadherin. Proteins comprising the transmembrane domain localized to the membrane fraction. Deletion of this domain resulted in a predominantly nuclear localization of the cytoplasmic segment of hFat1. Nuclear localization was largely reduced by deletion of a presumed juxta-membrane NLS. Fusion proteins located in the plasma membrane underwent proteolytic processing. In a first proteolytic step, only the extracellular domain was cleaved off. In another step, the cleavage product was released to the cytosol and was also found in a low speed pellet fraction, in accordance with the nuclear localization of the cytoplasmic domain of hFat1. D 2005 Elsevier Inc. All rights reserved. Keywords: Protocadherin; Intramembrane proteolysis; Nuclear translocation; RIP; Development

Introduction Human Fat1 protein (Footnote 1) is a class I transmembrane protein and a nonclassical member of the cadherin superfamily consisting of more than 4500 amino acids [1,2]. The extracellular portion of this giant protein contains 34 cadherin-like repeats. Unlike classical cadherins, these repeats are complemented in hFat1 by a laminin A – G domain and by five potentially Ca2+-binding EGF-like domains, as evaluated by the program SMART [3]. Amino acids 4184 –4204 correspond to the transmembrane segment that is followed by a C-terminal intracellular domain of 386 amino acids. Although the overall sequence of this domain is in general not closely homologous to the cytoplasmic domains of classical cadherins, it shares minor similarities in Abbreviations: a.u., arbitrary units; CFG, cadherin-Fat-EGFP chimera; CTF1, CTF2, C-terminal fragment 1, 2; NLS, nuclear localization sequence; PBS, phosphate-buffered saline solution; PMA, phorbol 12myristate 13-acetate; PMSF, phenylmethylsulphonyl fluoride. * Corresponding author. Fax: +49 7531 88 2903. E-mail address: [email protected] (H.W. Hofer). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.03.006

regions which are possibly important for cadherin-like function, like catenin binding and anchoring to the cytoskeleton [4]. However, it is not known whether the Fat protein exerts these functions. The fat gene was originally discovered in Drosophila, where it is regarded to function as a tumor suppressor gene [5]. Its deletion causes hyperplastic over-growth of larval imaginal discs, defects in differentiation and morphogenesis, and lethality at the pupal stage [6]. Another protein of the Drosophila Fat family, mainly expressed in luminal organs, is encoded by the ftl locus and is more closely related to three homologous Fat proteins (Fat1, 2, and 3) in humans [7] whereas the putative human Fat4 protein appears more homologous to Drosophila Fat. Fat1( / ) mice exhibit perinatal lethality, loss of renal glomerular slit junctions, and partially penetrant anophthalmia and holoprosencephaly [8], i.e., defects that also indicate the importance of the protein for development and organ integrity. The study of molecular mechanisms involved in the biological functions of Fat is impeded by the enormous size of the extracellular moiety of the protein. In a first approach

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to circumvent this problem, we replaced the extracellular domain of human hFat1 by the extracellular domain of Ecadherin. In addition, expression vectors were generated that were deleted of the cadherin sequence but maintained the hFat1 intracellular domain. Expression of the chimeric proteins in cultured cells revealed partial shedding of the ectodomain, cleavage of the cytoplasmic segment from the transmembrane domain, and its ability to translocate to the nucleus. Intramembranous processing has been previously described for several other class I transmembrane proteins, like APP [9], Notch [10], ErbB4 [11], CD44 [12], and Ecadherin [13]. Two separate proteolytic events have been shown to be typically involved: first, processing of the ectodomain by a membrane-associated metalloprotease of the ADAM family, followed by a cleavage in the transmembrane region exerted by the g-secretase complex [14]. The data now reported suggest that the processing of hFat1 might follow similar pathways.

Materials and methods Plasmid constructs cDNA of the cytoplasmic domain of human Fat1 [4] was introduced into BglII/SalI sites of pEGFP-C1 (Clontech) using the primers 5V-GCAGATCTATCGTAAGATGATTAGTCGG-3V and 5V-CGCTGGACTCAGACTTCCGTGTGCTGCTG-3V. Deletion of the putative nuclear localization sequence was performed using the forward primer 5V-GCAGATCTCATCAGGCTGAACCTAAAGA3V. cDNA of murine E-cadherin was kindly provided by Dr. Nagafuchi [15] subcloned into BglII/SalI sites of pEGFP-N3 (Clontech) using the primers 5V-GCAGATCTATGGGAGCCCGGTGCCGCA-3V and 5V-CGCTCGAGCTAGTCGTCCTCGCCACCGC-3V. The E-cadherin/ Fat chimera (CFG) was generated using PCR and subcloned into BglII/SalI sites of pEGFP-N3. Primers were P1: 5VGCAGATCTATGGGAGCCCGGTGCCGCA-3V and P2: 5VAGGAACTTGCAATCCTGCTGC-3V for the extracellular portion of murine E-cadherin and the primers P3: 5VAGCAGGATTGCAAGTTCCTGGAATTGGAATCGTTGTGT-3V and P4: 5V-CGGTCGACGACTTCCGTGTGCTGCTGGGA-3V for transmembrane and cytoplasmic parts of hFat1. Amplified fragments using primer combinations P1/P2 and P3/P4 were used in a second amplification step with the primer combination P1 and P4 to generate the CFG chimera. Likewise, DE-CFG was produced using the primers 5V-GCGGGAATCGTGGCAG CAGGA-3V and 5VTGCCACGATTCCCGCTCGTTTCTGTCTTCT-3V instead of P1 and P2. Antibody production hFat1-c (fused to a H6-tag at the C-terminus) was expressed in E. coli, purified to electrophoretic homogeneity

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by chromatography on a Ni-agarose column, and used for immunization. Monospecific antibodies were prepared from the rabbit antiserum according to the method described by Olmsted [16]. Cell culture and transfections Human embryonic kidney cells 293 (HEK293) and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 2 mM l-glutamine. HEK293 cells were transfected using Fugene 6 (Roche) and transfection of HeLa cells was carried out using Lipofectamine2000 (Invitrogen). Phorbol 12-myristate 13-acetate (PMA) was dissolved in Me2SO and applied at a final concentration of 1 AM. Microscopy For confocal microscopy, 2.5  105 cells were plated on glass coverslips in 6-well plates (< 20 mm) 24 h prior transfection. Between 14 and 24 h after transfection, cells were fixed in 3.7% formaldehyde, washed with PBS (5 times), and mounted in Aqua-Poly/Mount (Polysciences). For immunostaining, fixed cells were permeabilized prior to mounting with 0.1% Triton X-100 in PBS (10 min), washed with PBS (5 times), and incubated with antibodies obtained from the pre-immune or immune serum (1:200 in PBS plus 1% low-fat milk powder) for 1.5 h at room temperature followed by washings with PBS (5 times) and incubation with Cy3-conjugated anti-rabbit IgG (1:1000 in PBS plus 1% milk powder, 1 h). Laser-scanning confocal microscopy was done with a LSM510 microscope on an inverted stand using the Plan-Apochromat 63/1.40 objective (Carl Zeiss MicroImaging). Western blot analysis Cells were lysed in Laemmli buffer 24 h after transfection. Samples were separated on 8% SDS –PAGE gels and transferred to nitrocellulose membranes for 1 h at 40 mA/cm2 using a semi-dry transfer system. The membranes were blocked with 1% BSA in Tris-buffered saline for 1 h and probed with a monoclonal GFP antibody (Roche, Cat. No. 1814460). Anti-mouse horseradish peroxidase-conjugated secondary antibody (Calbiochem) was used followed by visualization with ECL luminescence kit (Amersham). Subcellular fractionation Cells (5  106) were collected in lysis buffer (50 mM Tris – HCl pH 7.4, 10 Ag/ml aprotinin, 40 AM PMSF and 1 mM EDTA), lysed by passing through a 27 Gx3/4-in. needle, and then centrifuged at 1000  g for 1 min. The pellet was washed twice in lysis buffer and used as ‘‘nuclear’’ fraction. It did not contain activity of phosphofructokinase nor aldolase, but was the only fraction

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containing DNA. The supernatant was centrifuged again at 170,000  g (30 min) to separate into soluble and membrane fraction. The soluble fraction (‘‘cytosol’’) contained all phosphofructokinase and aldolase activity. In vitro proteolysis Transiently transfected HeLa or non-transfected HEK293 cells were washed twice with PBS. Next, the cells were suspended in hypotonic buffer (10 mM PIPES pH 7.4, 10 mM KCl, 2 mM MgCl2) and incubated 30 min on ice followed by homogenization in a Dounce glass homogenizer (30 strokes). The disrupted cells were centrifuged at 200  g for 10 min to remove unbroken cells and cell nuclei. The supernatant from HeLa cells (800 Al) was mixed with the supernatant from HEK293 cells (200 Al) and incubated in the presence or absence of a protease inhibitor (g-secretase inhibitor IX, 1 AM, or pepstatin1 Ag/ml) or DMSO (1 Al/ml as control) at 37-C for 4 h. The incubation mixtures were cleared by centrifugation at 130,000  g for 1 h (Beckman TLA 100.2 rotor, 55.000 rpm). The supernatants were analyzed by immunoblotting after immunoprecipitation with anti-GFP-antibody (Roche). The variation of the Ponceau staining of the IgG heavy chain band was within T1% (loading control). Densitometric evaluation was performed with the Quantity One program (BioRad).

Results

Fig. 1. Summary of the hFat1-EGFP fusion constructs used in this report. (A) Structure of the CFG chimeric protein. (B) Structure of the DE-CFG protein devoid of the extracellular E-cadherin domain. (C) Structure of the c-FG fusion protein comprising only the cytoplasmic hFat1 domain. (D) Structure of the DNLS-c-FG fusion protein deleted of the putative nuclear localization sequence of the cytoplasmic hFat1 domain.

Construction and localization of chimeric hFat1 To circumvent the technical problems derived from the unusually large extracellular domain of hFat1, we constructed expression vectors encoding shorter transmembrane proteins fused to the EGFP marker. The first vector codes for a chimera between the extracellular domain of pre-proE-cadherin and the transmembrane and intracellular domains of hFat1 (Fig. 1A). The cytoplasmic moiety of this chimera (abbreviated as CFG for cadherin, Fat1, and GFP) contained the EGFP marker fused to the C-terminus.

The second vector, named DE-CFG, was deleted for the extracellular E-cadherin sequence but still contained the preand pro-sequences of E-cadherin, the trans-membrane, and intracellular sequences of hFat1, and EGFP. A vector constructed to express E-cadherin linked to EGFP served as control. E-cadherin fused to EGFP and the hFat1 chimera were successfully expressed in HEK293 cells as proven by immunoblotting and fluorescence microscopy (Figs. 2A – C). About 40% of the cells were transiently transfected by

Fig. 2. Confocal images of the localization of E-cadherin and of CFG chimeric protein. Cells were cultured, transfected, and processed as described in Materials and methods. (A – C) Localization in HEK293cells of (A) E-cadherin-GFP, (B) CFG, and (C) DE-CFG. Observe the delineation of the nuclear and plasma membranes as well as the unlabeled nuclei. The focal cytoplasmic accumulation of labeled hFat1 might be related to the ‘‘fibroblastoid migratory’’ morphology of the cells (scale bars 10 Am). (D – G) Localization in HeLa cells of (D) E-cadherin-GFP, (E) and (G) CFG, (F) DE-CFP. Observe the uniform granular cytoplasmic distribution of E-cadherin with completely unlabeled nuclei (D), the concentration of the CFG fusion protein at lamellipodia (E), and the labeling of the nucleoplasm but free nucleoli (F and G), and speckle-like aggregates (G) (scale bars 10 Am). Fig. 3. Nuclear localization of hFat1. (A – D) Nuclear localization of c-FG (A and C) and DNLS-c-FG (B and D) in HEK293 (A and B) and HeLa (C and D) cells. Cells were cultured, transfected, and processed as described in Materials and methods. Note that c-FG protein localizes preferentially to the nucleus with some aggregation in ‘‘speckles’’ but is absent from the nucleoli (A and C). (Scale bars 10 Am.) (E – G) Comparison of GFP fluorescence (F) in c-FG-transfected HeLa cells with the immunostaining by the antiserum against the cytoplasmic domain of hFat1 (E). (H – J) Staining of endogenous hFat1 in HeLa cells with the antiserum against the cytoplasmic domain of hFat1 (H) and simultaneous staining of the nuclei with Hoechst 33342 (I). Note the absence of hFat1 in the nucleoli. (K) Immunostaining of hFat1 in a 0.4-Am serial Z-axis section through the middle of a single cell (section 23 out of 50). (L) Overlay of the staining with Hoechst 3342 in the same section. (M) Scan of the staining intensities along the line shown in L. (A movie showing the z-axis stacking is available as supplementary material.)

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Fig. 2.

the method used. The localization of E-cadherin-EGFP (Fig. 2A), the CFG chimera (Fig. 2B), and the DE-CFG construct (Fig. 2C), all expressed with the pre- and pro-sequences of

E-cadherin, were similar in HEK293 cells. Significant amounts of the proteins were present in cytoplasmic granules and along cytoskeletal structures but the proteins

Fig. 3.

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were absent from the nuclei. This localization of the fusion proteins was similar to that recently described for pre-proN-cadherin [17]. Expression of the CFG chimera in HeLa cells resulted in a different localization compared to that in HEK293 cells (Fig. 2E) while E-cadherin was similarly distributed in both cell types (Figs. 2A and D). The pattern observed in about 95% of the CFG-transfected HeLa cells (transfection efficiency about 60%) was of clusters of fluorescent protein accumulated in or near the basis of lamellipodia (Figs. 2E and F). Only minor amounts were observed as cytoplasmic granules or vesicles. The localization of the DE-CFG polypeptide containing the cytoplasmic and transmembrane domains of Fat1 was similar to that of the CFG chimera. The highest concentration of fluorescent protein was also found in the lamellipodia (Fig. 2F) and moderate fluorescence was observed in the cytoplasm and in the nucleus. Conspicuous nuclear localization was observed in about 4% of the HeLa cells expressing the CFG chimera (Fig. 2G). In these cells, the lamellipodia were also labeled and some protein appeared localized to cytoplasmic vesicles. The nuclear fluorescence spared the nucleolus, and in a number of these cells the protein appeared concentrated in dot-like structures resembling ‘‘speckles’’ [18] (Fig. 2G). Localization of the cytoplasmic domain of hFat1 in HEK293 and HeLa cells The cytoplasmic domain of hFat1 expressed both in HEK293 and in HeLa cells as fusion protein with enhanced green fluorescent protein (construct c-FG, see Fig. 1D) was almost exclusively localized to the nucleus (Figs. 3A and C). The plasma membrane, however, did not show the modest fluorescence observed with CFG transfectants. A nuclear localization was also observed for c-FG-transfected L- and CHO cells, and cell lines exhibiting stable expression of cFG (not shown). The intense nuclear staining was diffuse and clearly delineated the non-fluorescent nucleoli (Figs. 3A and C). In 20 – 30% of the transfected cells, a significant amount of fluorescent protein was concentrated in speckle-like structures (inserts in Figs. 3A and C), as observed also for about 4% of CFG-transfected HeLa cells (Fig. 2G). Inspection of the amino-acid sequence of the cytoplasmic domain of hFat1 revealed clusters of basic amino acids near the N-terminal end that we interpret as nuclear localization sequences (NLS). This conclusion was supported by the results showing that deletion of the nucleotides encoding the amino acid sequence RKMISRKKKH (amino acids 4204– 4214 of hFat1, construct DNLS-c-FG, cf. Fig. 1D) led to an intense cytoplasmic staining in HEK293 and somewhat less in HeLa cells. Although still some fusion protein localized to the nucleus, extranuclear localization was distinctly enhanced (Figs. 3B and D). The distribution and localization of the NLS-deleted protein were clearly different from that of EGFP which distributed evenly in the transfected cells (not shown).

Immunocytochemical localization of endogenous hFat1 Localization of endogenous hFat1 was studied in HeLa cells (as well as in HEK293 and HaCat [19] cells, data not shown) using the polyclonal antiserum raised against the cytoplasmic domain of hFat1 expressed in bacteria. The specificity of the immunoreaction was proven by the colocalization of immunofluorescence and GFP fluorescence of c-FG overexpressed in HeLa cells (Figs. 3E – G). The merged image (Fig. 3G) demonstrates complete colocalization of immuno- and GFP fluorescence of the protein in the nuclei and absence of c-FG from the nucleoli. Immunostaining of the cytoplasm and the plasma membrane was visible similar as in cells not over-expressing hFat1 constructs (see below). Samples treated with the preimmune serum only exhibited background fluorescence. The specificity of the immunoreaction was further supported by the fact that the images also showed cells whose nuclei did not react with anti-c-FG and was also proven by comparison of immunoblots of extracts from cells overexpressing the various hFat-c constructs probed with antiGFP and the antiserum. Partial nuclear localization of hFat1, analogous to that depicted for HeLa cells, was also observed in HEK293 and HaCat cells (not shown). Fig. 3H shows the localization of endogenous hFat1 in HeLa cells as revealed by confocal microscopy (0.8-Am section, thickness of the nuclei about 6.5 Am). Immunoreactive hFat1 was detected on the plasma membrane, in the perinuclear space of the cytoplasm, and, notably, also in the nuclei of a substantial number of cells. Nuclear localization of hFat1 in these cells was confirmed by co-staining with Hoechst 33342 (Figs. 3I and J) and by Z-stacking images. Fig. 3K represents such a 0.4-Am section through the middle of a single cell stained with the antiserum. Endogenous hFat1 is visible in the nucleus and in the perinuclear zone as well as in the plasma membrane. Fig. 3L shows the overlay of Hoechst 33342 staining and indicates the level of the intensity scan shown in Fig. 3M for the same cell. This scan proves co-localization of the immunostaining (red trace) with DNA staining (blue trace) in the nucleus. Spontaneous proteolytic cleavage of hFat1 constructs comprising the transmembrane sequence The nuclear localization of the cytoplasmic moiety of hFat1 and the nuclear localization of fluorescent protein in some CFG-transfected HeLa cells (Fig. 2G) stimulated the hypothesis of a proteolytic release and nuclear translocation of the cytoplasmic moiety of the CFG protein. Fig. 4A shows a protein blot of extracts from CFG-transfected cells immunodetected with monoclonal anti-GFP. Extracts from HEK293 cells exhibit a broad band corresponding to the CFG chimera (Fig. 4A, left lane). Short exposition of the blots (not shown) revealed two separate bands which may correspond to the primary expression product (the pre-pro-

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Fig. 4. Processing of the CFG and DE-chimeras of hFat1 in HEK293 (left lanes) and HeLa (right lanes) cells. Blots of extracts after SDS – PAGE of cells transfected either with the CFG chimera (A) or its derivative DE-CFG lacking the E-cadherin extracellular domain (B) were probed with anti-GFP. The positions of CFG and DE-CFG are indicated. The asterisk marks the position of the main expression product after transfection with a vector encoding the pre-pro-form of DE-CFG. The bands marked as CTF1 and CTF2 correspond to cleavage products of different size whose comparison is facilitated by dotted lines.

form of CFG) and a processed form. Note that in extracts from HeLa cells a smaller second band appeared (designated CTF1, Fig. 4A, right lane) in addition to the high-molecular weight bands. Fig. 4B shows the separation of extracts from cells transfected with the DE-CFG construct that comprises the amino acids of the pre-pro peptide sequences (aa 1– 156) and aa 700– 710 of E-cadherin, and the transmembrane and

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cytoplasmic domains of hFat1 fused to EGFP. The electrophoreses show three major bands reacting with anti-GFP. The strongest (top) band is likely to correspond to the primary expression product (including the pre- and pro-sequences). The migration of the middle band corresponds to that of the CTF1 fragment of Fig. 4A. The lower band (named CTF2) did not appear in expression experiments with the full-length CFG chimera. The hFat1 protein lacking the extracellular domain is, therefore, efficiently processed both in HeLa and HEK293 cells. It appears that proteolysis is to some extent progressive in HEK293 cells (Fig. 4B, left lane). To determine the cellular localization of the different polypeptides, extracts of HEK293 cells expressing the various hFat1-derived constructs were fractionated by differential centrifugation and analyzed by SDS – PAGE and antiGFP immunoblotting (Fig. 5A). The high-speed pellet fraction, assumed to contain plasma and intracellular membranes (Fig. 5A, left panel), contains the CFG polypeptide and the DE-CFG chimera deleted of the E-cadherin moiety. Two forms (corresponding to the two upper bands in Fig. 4B) of the DE-CFG protein are present in this fraction. They may represent the primary expression product and a processed form of the protein. The membrane fraction is free of the cytoplasmic forms (with and without nuclear localization sequence, middle panel). The 100,000-g supernatants (cytosol, Fig. 5A, middle panel) do not contain the forms

Fig. 5. Fractionation of extracts from HEK293 (A) and HeLa (B) cells expressing hFat1 constructs. Cells were transiently transfected with either CFG, DECFG, c-FG, or DNLS-c-FG as indicated on the top of the figures. Blots of extracts of cellular fractions were probed with anti-GFP after SDS – PAGE. The positions of CFG and DE-CFG are indicated. The asterisk marks the position of the main expression product after transfection with a vector encoding the prepro-form of DE-CFG. The bands marked as CTF1 and CTF2 correspond to cleavage products of different size. Note that CTF1 resides in the membrane fraction and is absent from the cytosol. CTF2 is not detected in the membrane fraction but is present in the cytosol. Experiments A and B were evaluated by two separate blots.

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detected in the membrane fraction, but evidently contain a proteolytic cleavage product of DE-CFG exhibiting the same migration as the peptide termed CTF2 in Fig. 4B. The size of CTF2 is slightly larger than that of c-FG, also present in the cytosol. The slight differences in electrophoretic mobilities between CTF2, c-FG, and DNLS-c-FG correspond to differences in molecular weight of about 1000, allowing for the conclusion that the CTF2 cleavage product comprises an extension of 5– 10 amino acids into the N-terminal transmembrane sequence when compared to c-FG. The third fraction documented in Fig. 5A (right panel) represents the pellet of low speed centrifugation and contains nuclei as well as membranes. Accordingly, the components observed in the plasma membrane fraction are also present. No cleavage product was present in this fraction when the CFG chimera was expressed in HEK293 cells, whereas the CTF2 polypeptide generated in the DE-CFG experiments was present while it was absent from the membrane fraction. The low speed pellet also contained the c-FG expression product but was almost free of DNLS-c-FG. An analogous separation of HeLa extracts (Fig. 5B) showed minor differences to that of HEK293 cell extracts. In addition to uncleaved CFG polypeptide, the membrane fraction (left panel) contained a cleavage product, corresponding to CTF1 in Fig. 4A. This polypeptide is smaller than the DE-CFG construct (upper band and even slightly smaller than the lower band), suggesting that CTF1 was devoid of the ectodomain. The cytosol fraction was free of CTF1, suggesting that CTF1 was still anchored to the membrane and may have retained elements of the transmembrane domain. The cytosol of cells transfected with the DE-CFG construct contained a different cleavage product, smaller than CTF1, and corresponding in its size to CTF2 of Fig. 4B. A very small amount of this fragment was also present in the low speed pellet (Fig. 5B, right panel). These results indicate that the HEK293 cells were unable to cleave the ectodomain of the CFG-chimera but were able to process the protein that did not comprise this ectodomain and to release the product into the cytosol and into intracellular structures. In contrast, the HeLa cells had the ability to cleave off the ectodomain of the CFG chimera. According to the size of this product and since it remained in the membrane, the cleavage presumably took place at the outer side of the membrane. Further cleavage of the CFG chimera and release of the product into the cytosol were not prominent in HeLa cell fractions although nuclear localization of fluorescent fusion protein was observed in about 4% of transfected cells. However, some cleavage to the size of CTF2 and release to the cytosol occurred after DE-CFG transfection, but was less than that observed with HEK293 cells. In vitro cleavage of the CFG chimera The requirement of two different proteolytic systems, one mainly active in HeLa cells, the other predominantly active in HEK293 cells for the cleavage of the CFG chimera, was

supported by the results of the in vitro experiments employing extracts from both cell lines (cf. Materials and methods). The CFG chimera was expressed in HeLa cells and the cell homogenate was supplemented either with buffer or HEK293 cell homogenate. GFP fusion proteins were immunoprecipitated and detected with anti-GFP on blots after separation of the precipitates by SDS – PAGE. This was followed by the densitometric evaluation of the intensity of the band corresponding to CTF2. Only a small amount of this polypeptide was present in the experiments without HEK293 homogenate (17 T 0.9 a.u.), whereas there was a 3.7-fold increase when HEK293 homogenate was added (63 T 3 a.u.). The intensity of the band was not affected by the addition of a small amount of DMSO (63 T 0.5 a.u.). The appearance of the band, however, was distinctly inhibited when the aspartate protease inhibitor pepstatin was present in the experiments (29 T 2 a.u.) and even more by the presence of the g-secretase inhibitor IX (22 T 2 a.u.).

Discussion The fat (ft) locus in Drosophila [6], a member of the cadherin superfamily [1], is essential for certain developmental processes and is also considered to be a tumor suppressor gene [5], but the mechanisms of its action have remained elusive. Here we report data for the human homologue of the Fat-like human protein hFat1 that support a novel signaling mechanism from the plasma membrane to the nucleus employing the cytoplasmic domain of the hFat1 protein. Although hFat1 is a class I transmembrane protein like most members of the large cadherin family of receptors, the preferred localization of the isolatedly expressed cytoplasmic domain of hFat1 in fusion with EGFP (termed c-FG in this report) was nuclear. This result is in agreement with the presence of the sequence motif RKMISRKKKH close to the N-terminus of the cytoplasmic domain, which is indeed predicted to be an exemplary nuclear localization sequence. Deletion of this sequence distinctly changed the localization pattern and increased the amount of extranuclear protein, suggesting its importance for nuclear translocation. However, additional domains may also be involved in the determination of nuclear localization. Within the nucleus of many transfected cells, the cytoplasmic domain of hFat1 exhibited a conspicuous focal localization reminiscent of nuclear speckles [18]. It is unclear whether this results from the overexpression of the protein or provides a hint for a potential function in the nucleus as reported for speckle proteins. A recent report described physical interactions of Drosophila Fat with the nuclear protein atrophin [20]. To be of functional relevance, this would also require nuclear translocation of the cytoplasmic domain. In mammals, atrophin-1 and -2 are transcriptional repressors that interact with the histone deacetylases [21,22].

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We used a chimeric construct consisting of the extracellular domain of E-cadherin and the transmembrane and cytoplasmic domains of hFat1 to study mechanisms which may be involved in such translocation. This artificial system avoided problems inherent with the heterologous expression of the huge extracellular domain of hFat1 [4] encoded by about 15 kilobases. The similarity of the results obtained with two human cell lines of widely different origin and biological characteristics (HeLa = cervix carcinoma; HEK293 = embryonal kidney) indicates that the results are not cell- or tissue-specific. The localization of the chimeric construct expressed in HEK293 cells was exclusively extranuclear and included a discrete labeling of the plasma membrane. In transfected HeLa cells, the chimera was prominently concentrated in lamellipodia in the same manner as recently described for Fat1 by Tanue and Takeichi [23]. Since the localization pattern of EGFP-fused E-cadherin was different in HeLa cells, the extracellular Ecadherin moiety of the CFG chimera appears not to be a strong determinant of its subcellular localization. A minor but significant fraction of transfected HeLa cells exhibited fluorescent protein in the nuclei (Fig. 2G) in the same focal arrangement as observed in cells expressing the transfected cytoplasmic domain (Fig. 3C). Apparently, some of the HeLa cells were able to release the cytoplasmic domain from the plasma membrane, although only a membraneanchored cleavage product (termed CTF1) was detected in the cell fractionation experiments (Fig. 5B). The failure in detecting a soluble or nuclear cleavage product is likely to result from the limited sensitivity of the immunoblot reaction and, possibly, also rapid degradation. The presence of endogenous hFat1 was demonstrated in HeLa cells by the immuno-reaction with an antiserum raised against the intracellular domain. The majority of the protein was detected in the cytoplasm and in the plasma membrane. These data were in good correspondence with the morphologic distribution patterns observed with over-expressed CFG constructs. A smaller, but nevertheless distinct, reaction also occurred in the nuclei of a number of cells, except of mitotic cells, as documented by confocal laser scanning microscopy. The staining pattern and localization of endogenous hFat1 corresponded very well with that recently published by [23]. In addition to the CFG chimera, we used a shortened form (DE-CFG) devoid of the extracellular E-cadherin domain, but still including the pre- and pro-sequences of E-cadherin. This construct may mimic the result of an extracellular cleavage event. When expressed in HEK293 or HeLa cells, the shortened, E-cadherin-deleted DE-CFG protein exhibited the same localization as the CFG chimera. Different from the CFG-chimera, however, the DE-CFG protein was cleaved not only in HeLa but also in HEK293 cells, yielding a product (termed CTF2) that was slightly smaller and, more importantly, was released to the cytosol. The comparison of the electrophoretic mobility of CTF2 with those of c-FG and DNLS-c-FG indicates a molecular

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mass about 1000 Da higher than that of c-FG, corresponding to a N-terminal elongation of 5– 10 amino acids into the transmembrane domain. Proteolytic processing of class I transmembrane proteins appears to occur frequently and has been found to be involved in signal transduction. This is typically a two-step process requiring the successive action of an extracellular metalloprotease and an intracellular protease system acting in or at the cytoplasmic side of the plasma membrane [14]. HEK293 cells did not cleave the CFG chimeric protein containing the extracellular cadherin sequence but were able to process the protein deleted of it. The simplest explanation for this result is the assumption that these cells lack an active metalloprotease required for the shedding of the ectodomain and that this step is a prerequisite for the second cleavage. This assumption is supported by the finding that shedding of the ectodomain was mimicked by its omission in the DE-CFG construct, thus allowing an intracellular protease to cleave the protein. Since only a fraction of the expressed DE-CFG protein was localized to the plasma membrane, the cleavage may have taken place in intracellular compartments. HeLa cells were evidently able to perform an extracellular cleavage of the CFG chimera resulting in a C-terminal cleavage product still residing in the membrane fraction. As shown by Fig. 3, however, this apparently spontaneous release is infrequent and may be triggered by yet unknown signals or internal events. Stimulation of the proteolytic cleavage of the CFG chimera by PMA (data not shown) already described for Notch [10] and CD44 [24] suggests modulation by extracellular stimuli. However, cleavage evidently occurred at the outer side of the plasma membrane and did not result in the release of a soluble product. The difference in the data obtained with HeLa and HEK293 data is assumed to be due to the capacity of HeLa but not HEK293 cells to cleave the cadherin domain from the CFG chimera. HEK293 cells, however, were able to release the intracellular segment from the transmembrane domain when the cadherin segment had been previously removed. The conclusion is further supported by in vitro experiments with the CFG chimera expressed by HeLa cells. The release of the intracellular CTF2 domain was low in the HeLa cell homogenates, but increased distinctly after the addition of HEK293 cell homogenate. The proteolytic event was inhibited by low concentrations of the g-secretase inhibitor IX and by pepstatin, which is also an inhibitor of g-secretase. The protocadherin hFat1 is involved in signaling processes both during development [8] and also of importance in mature organisms [25]. It appears to regulate cell polarity as well as cell –cell adhesion. Like other members of the cadherin family, it may participate in the control of cytoskeletal interactions and in actin polymerization [23]. The observations presented here provide evidence for an additional mechanism that involves regulated intramembranous processing and nuclear targeting of

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the cytoplasmic domain. This mechanism would distinguish hFat1 from the well-analyzed catenin signaling system of classical cadherins [26].

[10]

Acknowledgments [11]

We are indebted to Dr. A. Nagafuchi for the kind gift of cDNA clones encoding the pre-pro form of E-cadherin, to Dr. W. Nastainczyk (Homburg/Saar) for expert support with the preparation of the antiserum, and to Dr. E. Ferrando-May for generous help with confocal microscopy. We thank Dr. Eneida Franco Vencio for valuable support with cell culture and Dr. Alexandra Porsche for the hFat1 clone used for the study. T.M. is a recipient of a stipend by the Studienstiftung des Deutschen Volkes and G.P.S. is supported by the German Academic Exchange Service (DAAD).

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2005. 03.006.

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