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1 Division of Biochemistry and 2 Central Service Unit, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Aichi 464-8681, Japan
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Introduction |
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Recent studies have shown that the LRR domains of LAP proteins mediate their basolateral membrane localization in epithelia (Legouis et al. 2003; Zeitler et al. 2004; Navarro et al. 2005; Quitsch et al. 2005). Legouis et al. reported that the LRR domains of C. elegans LET-413 and human ERBIN, but not the PDZ, are essential for the basolateral localization of these proteins in epithelial cells (Legouis et al. 2003). In addition, they revealed that the LRR domain but not the PDZ domain is necessary for LET-413 to function during C. elegans embryogenesis (Legouis et al. 2003). It was reported that the LRR domain of Drosophila Scribble tethers the protein to the plasma membrane, are both necessary and sufficient to organize a polarized epithelial monolayer, and are required for all proliferation control, whereas the PDZ domains, which recruit the LRR to the junctional complex, are dispensable for overall epithelial organization (Zeitler et al. 2004). Human Scribble was also reported to be restricted at the basolateral membrane of epithelial cells by its LRR domain, although constructs lacking LRR also showed some membrane localization (Navarro et al. 2005). The LRR region of human Densin-180 targets the protein to the basolateral membrane of transfected epithelial cells (Quitsch et al. 2005). Although it has become apparent, as described above, that proper basolateral localization is critical for LAP proteins to function, the molecular mechanism by which LAP proteins are recruited to plasma membranes remains unknown.
ERBIN was initially identified as a protein that interacted through its PDZ with ErbB2/HER2 receptor and was required for proper basolateral localization of the receptor (Borg et al. 2000). Thus far, many binding partners for ERBIN were reported, including PSD-95 (Huang et al. 2001), bullous pemphigoid antigen 1 (BPAG1) and Integrin β4 subunit (Favre et al. 2001), p120-retated catenins (Izawa et al. 2002b; Jaulin-Bastard et al. 2002; Laura et al. 2002; Ohno et al. 2002), SMADs (Warner et al. 2003), EBP50 (Rangwala et al. 2005), Nod2 (McDonald et al. 2005), Sur-8 (Dai et al. 2006) and Cav1.3 Ca2+ channels (Calin-Jageman et al. 2007). Although these interactions have provided us many clues to understand ERBIN functions, the molecular mechanism by which ERBIN regulates cell polarity remains unclear. In this study, we studied the mechanism of the plasma membrane targeting of human ERBIN. We found that ERBIN is palmitoylated at cysteines 14 and 16, and both palmitoylation and LRR are required for the plasma membrane targeting of ERBIN.
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Results |
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To further analyze the regulatory mechanism of ERBIN, we over-expressed various mutants of human ERBIN, carrying myc and polyhistidine (His) tags at the C-terminus, into HeLa cells and observed the intracellular localization (Fig. 1). Endogenous ERBIN localizes mainly at the plasma membrane of adherens junctions in HeLa cells (Izawa et al. 2002b), and in a single HeLa cell which does not have cell–cell contacts, the plasma membrane localization of endogenous ERBIN is not so marked (data not shown). When the wild-type (1–1371 amino acids-) ERBIN and 1–511 amino acids-ERBIN containing LRR were over-expressed in HeLa cells, both the wild-type and 1–511 amino acids-ERBIN localized mainly at the plasma membrane as previously reported (Legouis et al. 2003) (Fig. 1). In contrast, 33–511 amino acids-ERBIN did not localize at the plasma membrane, but was diffusely expressed in the cytoplasm, suggesting that the 1–32 amino acids of ERBIN are important for the plasma membrane localization (Fig. 1). Because 1–32 amino acids of ERBIN consist of the 22 amino acids which are located at the extreme N-terminus before the first LRR (LRR1) and a part of LRR1 (23–32 amino acids), this phenotype was supposed to be attributed to the lack of N-terminal 22 amino acids or LRR1. The 1–196 amino acids-ERBIN was localized to the cytoplasm, possibly due to the lack of the latter half of LRR. Changing the conserved proline residue within LRR into leucine (P315L), at a position equivalent to C. elegans LET-413 proline 305, also abolished the membrane localization of full-length ERBIN as previously described (Legouis et al. 2003), confirming that the LRR domain is necessary for the plasma membrane localization (Fig. 1). We also obtained the similar results in COS7 cells (data not shown). Taken together, these results encouraged us to further investigate the role of N-terminal 32 amino acids of ERBIN in the plasma membrane targeting.
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Based on the data described above, we first paid attention to the N-terminal amino acid sequences of ERBIN. The 1–32 amino acids of human ERBIN contains three cysteine residues at positions 14, 16 and 32 (Fig. 2A). In addition, other LAP family proteins, including human and rat Densin-180, human Scribble, Drosophila Scribble, and C. elegans LET-413, also contain some cysteine residues at N-termini (Fig. 2A). We next examined the subcellular fractionation of ERBIN. The subcellular fractionation of ERBIN was previously reported by two groups. Legouis et al. showed that ERBIN is found in Triton X-100-insoluble fractions in HeLa cells (18% of total ERBIN), as well as in the Triton X-100-soluble membrane fractions (21%) and the cytosol (61%), whereas in polarized MDCK cells, a smaller portion of ERBIN is cytosolic (39%) and the rest (61%) is recovered in the Triton X-100-insoluble fraction (Legouis et al. 2003). Navarro et al. showed the data demonstrating that in Caco-2 and MDCK cells, ERBIN is less present in the cytosolic fraction than in the Triton X-100-soluble or Triton X-100-resistant fraction (Navarro et al. 2005). To further characterize the association of ERBIN with particulate fraction of HeLa cells, we evaluated the extraction properties of the protein. HeLa cells were lysed with a homogenization buffer (HB) or HB supplemented with either 1% Triton X-100, 1 M NaCl, or 100 mM sodium carbonate buffer (SCB) (pH 11) (Fig. 2B). Western blot analysis showed that ERBIN is well extracted with nonionic detergent (1% Triton X-100) but poorly extracted with high salt (1 M NaCl) or high pH (SCB), displaying hydrophobic interaction of ERBIN with HeLa cell membranes (Fig. 2B). The presence of cysteine residues at the N-terminus as well as the hydrophobic partitioning of ERBIN in HeLa cells suggested the possible modification of the protein by fatty acid. As ERBIN lacks classical consensus sequences for N-terminal myristoylation or C-terminal prenylation (Johnson et al. 1994; Zhang & Casey 1996; Bijlmakers & Marsh 2003), we speculated that ERBIN protein might be palmitoylated. Accumulating evidence that protein palmitoylation, the post-translational modification of proteins with the lipid palmitate, is critical for the distribution of proteins to particular membrane locations (El-Husseini & Bredt 2002; Smotrys & Linder 2004) raised the possibility that palmitoylation of ERBIN may be crucial for regulating its trafficking and function.
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In the next step, we determined whether ERBIN protein was palmitoylated (Fig. 3). Immunoprecipitation of endogenous ERBIN from HeLa cells labeled with [3H]palmitate revealed that endogenous ERBIN is indeed palmitoylated (Fig. 3A).
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-myc polyclonal antibody. Western blotting showed that equivalent amounts of the wild-type and mutant ERBIN were expressed and purified from COS7 cells (Fig. 3B, lower panel). Fluorography showed that the mutation of either cysteine 14 or cysteine 16 or both almost completely eliminated palmitoylation of ERBIN (Fig. 3B, upper panel). In contrast, the mutation of cysteine 32 did not alter the extent of palmitoylation. These results showed that ERBIN is palmitoylated at cysteines 14 and 16 and mutating either cysteine 14 or 16 to serine completely disrupts palmitoylation of ERBIN. To determine if the [3H]palmitate was linked to ERBIN via a hydroxylamine–labile thioester linkage to cysteine residues, duplicate samples of labeled wild-type ERBIN purified from transfected COS7 cells were treated with hydroxylamine (NH2OH) or Tris–HCl buffer (Fig. 3C). Fluorography demonstrated that the incorporated [3H]palmitate was removed by hydroxylamine treatment, confirming that the incorporation of palmitate is due to the modification of cysteines through a thioester bond (Fig. 3C).
To test if other LAP proteins are also palmitoylated, COS7 cells transfected with myc-tagged human Densin-180 were labeled with [3H]palmitate, and myc-tagged Densin-180 proteins were isolated by immunoprecipitaion with -myc polyclonal antibody. Fluorography showed that myc-Densin-180 was palmitoylated (Fig. 3D). This indicates a possibility that palmitoylation may be a general post-translational modification in LAP family proteins.
Palmitoylation of ERBIN mediates association with plasma membranes
We next asked whether palmitoylation of ERBIN regulated association with plasma membranes. For this, we first checked if mutation of palmitoylated cysteine residues might affect the subcellular distribution (Fig. 4A). Transfected COS7 cells were lysed with a HB supplemented with 1 M NaCl to yield a soluble fraction (S) and an insoluble pellet (P). Palmitoylation-deficient mutants of ERBIN (S14C16C32, C14S16C32, S14S16C32, S14S16S32) were extracted with a high salt buffer while wild-type (C14C16C32) ERBIN and the C14C16S32 mutant that retained its palmitoylation remained particulate (Fig. 4A). These results demonstrated that the mutation of cysteines 14 and 16 induces the dissociation of the protein from membranes even if intact LRR is present.
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We next observed the intracellular localization of wild-type (C14C16C32) ERBIN and the palmitoylation-deficient mutant (S14S16C32) in polarized MDCK II cells (Fig. 4C). Wild-type (C14C16C32) ERBIN co-localized with β-catenin at basolateral membranes as previously described for endogenous ERBIN (Izawa et al. 2002b). In contrast, the palmitoylation-deficient mutant (S14S16C32) ERBIN, in which intact LRR is present, was expressed diffusely in the cytoplasm (Fig. 4C). We have examined the effects of the over-expression of the palmitoylation-deficient mutant (S14S16C32) ERBIN on epithelial polarity, but we have not been able to obtain a conclusive result by use of this over-expression system (data not shown).
Taken together, these results indicate that palmitoylation of ERBIN is necessary for the plasma membrane localization in HeLa cells and the basolateral membrane localization in polarized MDCK II cells.
Both palmitoylation and LRR are required for plasma membrane localization of ERBIN
We next examined the relationship between palmitoylation and LRR. For this, we determined if two ERBIN mutants, P315L and 1–196 amino acids (Fig. 1), were palmitoylated. COS7 cells transfected with wild-type (C14C16C32), P315L-, or 1–196 amino acids-ERBIN-myc-His constructs were labeled with [3H]palmitate, and subjected to immunoprecipitation with -myc antibody (Fig. 5). Western blotting showed that almost equivalent amounts of the wild-type and mutant ERBIN were expressed and purified from COS7 cells (Fig. 5, right panel). Fluorography showed that P315L-ERBIN was not palmitoylated, but 1–196 amino acids-ERBIN was palmitoylated (Fig. 5, left panel). These results indicate that a point mutation in LRR (P315L) inhibits the palmitoylation of ERBIN. In addition, because 1–196 amino acids-ERBIN lacks the latter half of LRR and localizes in the cytoplasm, it is suggested that the palmitoylation only is not sufficient for the plasma membrane localization, and the LRR domain is indeed necessary for the plasma membrane localization. These results suggest that both palmitoylation and LRR are required for ERBIN plasma membrane localization.
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To investigate if palmitoylation of ERBIN turns over in cultured cells, we examined the effects of 2-bromopalmitate, an inhibitor of protein palmitoylation, on ERBIN targeting on the plasma membrane in several cell lines (Fig. 6A). In a single HeLa cell, which did not have cell–cell contacts, the treatment with 2-bromopalmitate induced the marked loss of ERBIN localized at the plasma membrane, and increased the cytoplamic staining of ERBIN, compared to the staining of cells treated with control palmitate. In confluent HeLa cells, which contacted with adjacent cells, the treatment with 2-bromopalmitate did not result in the decrease in the plasma membrane localization of ERBIN (Fig. 6A). In MDCK II cells, which established firm cell–cell adhesions, the treatment did not apparently affect the ERBIN localization in cell staining. In non-epithelial cells that did not show marked cell–cell contacts, including U251MG, NIH3T3 and Neuro2A cells, the treatment with 2-bromopalmitate induced the marked translocation of ERBIN from the plasma membrane to the cytoplasm (Fig. 6A). The treatment with palmitate as a control did not affect the localization of endogenous ERBIN. We also examined the solubility of ERBIN in cultured cells treated with 2-bromopalmitate (Fig. 6B). After the treatment with 2-bromopalmitate or palmitate, HeLa, MDCK II and U251MG cells were homogenized and lysed in a HB supplemented with 1 M NaCl, and lysates were subjected to protein fractionation. The treatment with 2-bromopalmitate increased the amount of ERBIN in the soluble fraction (S) in these three cell lines, compared to that with palmitate. The increase of the solubility in non-epithelial U251MG cells was more apparent than that in epithelial HeLa and MDCK II cells, which was consistent with the results of cell staining as described above. These results indicate the dynamic turnover of palmitate on ERBIN at the plasma membrane. It is completely unclear at present why the effects of 2-bromopalmitate differed among several cell lines. It is tempting to speculate that the state of cell–cell contacts may affect the palmitate cycling on ERBIN.
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Discussion |
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We identified palmitoylation sites of ERBIN as cysteines 14 and 16 (Fig. 3). Mutating either cysteine 14 or 16 to serine almost completely disrupted palmitoylation and the plasma membrane localization of ERBIN (Figs 3 and 4). Some proteins have more than one palmitoylated cysteine. For example, PSD-95, a membrane-associated guanylate kinase, is dually palmitoylated at N-terminal cysteines 3 and 5, and mutating either cysteine 3 or 5 to serine completely disrupts palmitoylation of PSD-95 (Topinka & Bredt 1998; El-Husseini et al. 2000; Huang & El-Husseini 2005). Dual palmitoylation of PSD-95 mediates its vesiculotubular sorting, post-synaptic targeting, and ion channel clustering (El-Husseini et al. 2000). In addition, basolateral sorting of PSD-95 in polarized MDCK cells requires dual palmitoylation (El-Husseini et al. 2000). These characteristics of dual palmitoylation of PSD-95 are very similar to those of ERBIN.
LAP proteins other than ERBIN also contain N-terminal cysteine residues which are able to be palmitoylated (Fig. 2A). Navarro et al. performed fractionation on Caco-2 and MDCK cells, reporting that high amounts of human Scribble remained resistant to the Triton X-100 extracting buffer, evoking an association with the actin cytoskeleton or the lipid rafts (Navarro et al. 2005). Densin-180 was originally purified from the post-synaptic density fraction of rat brain, with the solubility profile that was consistent with anchoring of Densin-180 in the membrane fraction by a combination of lipid and protein interactions (Apperson et al. 1996). In this study, we observed that over-expressed Densin-180 was palmitoylated (Fig. 3D), suggesting a possibility that palmitoylation may be a general post-translational modification in LAP family proteins. Examination of the nature of the association of ERBIN with membrane fraction in HeLa cells indicated that ERBIN is associated with membranes through a combination of lipid and protein interactions (Fig. 2B). Since it is generally assumed that high pH buffers extract mainly peripheral membrane proteins, a substantial portion of ERBIN extracted with SCB (pH 11) is supposed to be a non-palmitoylated form (Fig. 2B). Thus, there may be both palmitoylated and non-palmitoylated pools of ERBIN, which is thought to be regulated by the balance of PAT and PTE activities. Palmitate turnover can be constitutive or regulated (Smotrys & Linder 2004). For example, it was shown that palmitate turnover on trimeric G protein subunit Gs is accelerated by its upstream β-adrenergic receptor (Wedegaertner & Bourne, 1994). It was also reported that glutamate receptor activity stimulates palmitate turnover on PSD-95, suggesting that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity (El-Husseini et al. 2002). In contrast to PSD-95 and Gs, palmitates on SNAP-25 are not dynamically cycled, and neuronal depolarization does not affect palmitate turnover on SNAP-25 (Kang et al. 2004). Together with other reports, these results indicate that palmitoylation can be dynamically regulated by specific extracellular signals, and the individual palmitoylation is regulated by both specific PATs and PTEs (Tsutsumi et al. 2008). Thus, the identification of specific PATs and PTEs of ERBIN and other LAP proteins as well as the search for possible factors regulating the balance of PAT and PTE activities will be necessary for further elucidation of the regulation of palmitoylation of these proteins.
As described earlier, the LRR domain has been believed to mediate a major part, if not all, of functions of LAP proteins (Legouis et al. 2003; Zeitler et al. 2004). On the other hand, two papers described the importance of Scribble PDZ domains as well as LRR for the localization and function (Albertson et al. 2004; Nagasaka et al. 2006). Albertson et al. found that both LRR and PDZ domains are important for the proper localization and function of Drosophila Scribble in neuroblasts (Albertson et al. 2004). In addition, Nagasaka et al. showed that the LRR and PDZ domain 1 are indispensable for human Scribble to inhibit cell growth by blocking cell–cycle progression and to keep its proper localization (Nagasaka et al. 2006). We and others previously reported that ERBIN interacts via its PDZ domain with p0071 (also called plakophilin-4), 
-catenin, and ARVCF, members of the p120 catenin family (Izawa et al. 2002b; Jaulin-Bastard et al. 2002; Laura et al. 2002; Ohno et al. 2002). We observed that ERBIN is associated with p0071, mainly at adherens junctions of epithelial cells (Izawa et al. 2002b). Ohno et al. observed that human ERBIN LRR as well as the middle and the C-terminal regions of ERBIN lacking LRR were localized at cell–cell contacts like the full-length ERBIN (Ohno et al. 2002). These data indicate that PDZ domains also play an important role in the plasma membrane localization of LAP proteins. In Fig. 6, we observed palmitate turnover on ERBIN in several cell lines, and noted that the turnover of palmitate on ERBIN in cells which form firm cell–cell adhesions might be slower than that in cells which do not have strong cell–cell adhesions. If this is the case, the recruitment of ERBIN to cell–cell adhesions through the PDZ domain and p0071 may affect the palmitate turnover on ERBIN.
It is intriguing that a point mutation in LRR (P315L) inhibits palmitoylation of ERBIN (Fig. 5). In addition, 1–196 amino acids-ERBIN that lacks the latter half of LRR is palmitoylated but shows a diffuse distribution. These results suggest that both palmitoylation and LRR are essential for proper localization of ERBIN, although the mechanism underlying the phenotype of these mutants is now completely unclear. The future study of the molecular function of LRR and the interplay of palmitoylation and LRR will help to dissect the process responsible for the establishment of apical–basolateral polarity and the formation of cell–cell junctions by ERBIN.
Palmitoylation promotes membrane association of otherwise soluble proteins. The function of palmitoylation, however, ranges beyond that of a simple membrane anchor (Smotrys & Linder 2004), including protein trafficking, G-protein signaling, ion channel clustering, post-synaptic plasticity and protein stability (El-Husseini & Bredt 2002; Smotrys & Linder 2004; Linder & Deschenes 2007). Recently, genetically conserved DHHC family proteins have emerged as PATs, and mutations of DHHC proteins in human are associated with various diseases, including cancers and neurological disorders (Tsutsumi et al. 2008). Although thus far we have been unable to demonstrate specific functions of the palmitoylation of ERBIN, the finding obtained in this study will provide a clue to further research on cell polarity and other signaling pathways regulated by ERBIN.
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Experimental procedures |
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HeLa, COS7, U251MG, NIH3T3, and Neuro2A cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin in an air-5% CO2 atmosphere with constant humidity. MDCK II cells were cultured in DMEM supplemented with 10% calf serum and penicillin/streptomycin. For cell fractionation, HeLa cells were disrupted in a HB, containing 50 mM Tris–HCl (pH 7.5), 5 mM EDTA, and 10 µg/mL leupeptin, by sonication on ice, and then extracted on ice for 30 min in either HB or HB supplemented with 1% Triton X-100, 1 M NaCl, or 100 mM SCB. Extracts were separated by centrifugation at 18 500 g into a soluble fraction (S) and an insoluble pellet fraction (P). Equal fractions were then analyzed by Western blotting with -ERBIN polyclonal antibody (Izawa et al. 2002b).
DNA constructions and transfections
KIAA1225 clone encoding human ERBIN cDNA was kindly provided by T. Nagase and N. Kusuhara (Kazusa DNA Research Institute, Chiba, Japan) and the full-length human ERBIN cDNA was obtained as previously described (Izawa et al. 2002b). Cysteine substitutions were generated by polymerase chain reaction (PCR)-based site-directed mutagenesis. Wild-type and mutant ERBIN constructs were subcloned into the pEF1/Myc-His vector (Invitrogen, Carlsbad, CA) to express C-terminal myc-polyhistidine (His)-tagged proteins. The mammalian expression vector encoding myc-tagged full-length human Densin-180 (pRK5-myc-Densin-180) was described previously (Ohtakara et al. 2002). COS7 and MDCK II cells were transiently transfected with LipofectAMINE 2000 (Invitrogen) as indicated by the manufacturer. HeLa cells were transiently transfected with LipofectAMINE PLUS (Invitrogen). Approximately 24 h after transfection, cells were processed as described for each experiment.
Immunofluorescence
Cells grown on coverslips were fixed in 50% methanol/50% acetone (vol/vol) at –20 °C for 10 min. To visualize myc-His-tagged proteins, cells were reacted with -myc monoclonal antibody (9E10), followed by Alexa 488-labeled -mouse antibody (Invitrogen). For double-staining for myc-His-tagged proteins and β-catenin, cells were first incubated with -myc polyclonal antibody (Sigma, St. Louis, MO), followed by Alexa 488-labeled -rabbit antibody (Invitrogen). Next the cells were incubated with -β-catenin monoclonal antibody (BD Biosciences, San Jose, CA), followed by FluoroLink Cy3-linked -mouse antibody (GE Healthcare, Piscataway, NJ). Confocal microscope systems (Radiance, Bio-Rad laboratories, Hercules, CA; LSM510, Zeiss, Thornwood, NY) were used to examine the coverslips.
Analysis of palmitoylation, immunoprecipitation and Western blotting
[3H]palmitoylation was performed essentially as described (Fukata et al. 2004, 2006). In brief, HeLa cells or transfected COS7 cells were pre-incubated for 30 min in serum-free DMEM with fatty acid-free bovine serum albumin (10 mg/mL; Sigma). Cells were labeled with 0.4 mCi/mL [3H]palmitate (31 Ci/mmol; PerkinElmer, Boston, MA) for 4 h in the preincubation medium. Cells were washed twice with PBS and lysed in a lysis buffer consisting of 1% Triton X-100, 20 mM Tris–HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 10 µM PMSF, 10 µg/mL leupeptin. Lysates were clarified by centrifugation at 18 500 g for 30 min. Endogenous ERBIN of HeLa cells was immunoprecipitated from cell lysates with -ERBIN rabbit polyclonal antibody (Izawa et al. 2002b) and myc-His-tagged proteins over-expressed in COS7 cells were immunoprecipitated with -myc rabbit polyclonal antibody (Sigma) for 1 h at 4 °C. Next, the samples were incubated with protein G-sepharose (GE Healthcare) at 4 °C for 1 h. The beads were then washed three times with the lysis buffer and boiled in SDS-PAGE sample buffer with 10 mM DTT for 2 min. For fluorography, protein samples were separated by SDS-PAGE. After fixing gels for 30 min in a fixing solution (isopropanol : water : acetic acid = 25 : 65 : 10), gels were treated with Amplify (GE Healthcare) for 30 min, dried under vacuum, and exposed to Kodak BioMax MS film at –80 °C for 7–14 days. For hydroxylamine treatment, duplicate gels were treated either 1 M Tris–HCl (pH 7.0) or 1 M hydroxylamine (pH 7.0) for 18 h at room temperature, and fluorographed as described above. For Western blotting, protein was electroblotted onto PVDF, probed with the appropriate antibody and horseradish peroxidase (HRP)-conjugated secondary antibody (GE Healthcare), and visualized using ECL-Plus reagent (GE Healthcare). To see the effects of blocking palmitoylation in cultured cells, cells were treated for 14 h with 100 µM 2-bromopalmitate or palmitate, and subjected to cell fractionation or immunofluorescence.
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Acknowledgements |
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Footnotes |
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* Correspondence: Email: iizawa{at}aichi-cc.jp
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References |
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Received: 20 December 2007
Accepted: 1 April 2008
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