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Address correspondence to Filippo G. Giancotti, Cell Biology Program, Memorial Sloan-Kettering Cancer Center, Box 216, 1275 York Avenue, New York, NY 10021. Tel.: (212) 639-6998. Fax: (212) 794-6236. email: f-giancotti{at}ski.mskcc.org
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Abstract |
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Key Words: keratinocyte; proliferation; palmitoylation; hemidesmosome; cysteine
L. Gagnoux-Palacios' present address is INSERM U385, School of Medicine, University of Nice, 06107 Nice, France.
M. Dans' present address is Department of Dermatology, University of Pennsylvania, Philadelphia, PA 19104.
W. van't Hof's present address is Athersys Inc., Cleveland, OH 44115.
A. Mariotti's present address is CePO/ISREC, 1066 Epalinges, Switzerland.
Abbreviations used in this paper: [125I]IC16, 16-[125I]iodohexadecanoic acid; EGF-R, EGF receptor; ERK, extracellular signalregulated kinase; HUVEC, human umbilical venous endothelial cell; PA-JEB, junctional epidermal bullosa with pyloric atresia; PI-3K, phosphatidylinositol-3 kinase; pSFK, palmitoylated Src family kinase; SFK, Src family kinase.
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Introduction |
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The lipid raftssubdomains of the plasma membrane enriched in cholesterol and glycosphingolipidspromote membrane compartmentalization of signaling components (Simons and Toomre, 2000). Because of the biophysical properties of their lipid anchor, GPI-linked receptors and palmitoylated signaling proteins, such as certain G proteins, H-Ras, many Src family kinases (SFKs), and eNOS, are concentrated in rafts (Resh, 1999). Here, we report that the 6ß4 integrin is incorporated in lipid rafts in a palmitoylation-dependent manner, and this is necessary to couple the integrin to a palmitoylated SFK (pSFK) and to promote mitogenic signaling.
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Results and discussion |
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The juxtamembrane segment of the ß4 tail contains a cluster of cysteines, which may be palmitoylated (Fig. 2 A). To examine this possibility, rat bladder 804G cells expressing a wild-type (A) or a tail-less (L) human ß4 were metabolically labeled with 16-[125I]iodohexadecanoic acid ([125I]IC16) palmitate analogue or [35S]methionine/cysteine and were immunoprecipitated with the antihuman ß4 mAb 3E1. Fig. 2 B shows that ß4 incorporated [125I]IC16, but 6 did not. Deletion of the ß4 tail prevented palmitoylation of ß4. In addition, treatment with alkali released the [125I] radioactive signal from ß4, implying that the radioactive palmitate analogue was attached to ß4 through a thioesther bond. Notably, ß4 was found to be palmitoylated to a higher apparent stoichiometry in HaCat keratinocytes (Fig. 2 B), primary human keratinocytes, and squamous carcinoma A431 cells (unpublished data). Although other integrin ß subunits do not contain potential palmitoylation sites, the cytoplasmic segments of
3,
6,
8, and
E contain one membrane-proximal cysteine. We did not detect any palmitoylation of
3ß1 and
6ß1 (unpublished data). Thus, ß4 may be the only integrin subunit modified by palmitoylation.
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To examine the role of lipid rafts in 6ß4 signaling to ERK, we transiently transfected vectors encoding various mutant forms of
6ß4 in ß4-negative human umbilical venous endothelial cells (HUVECs; Dans et al., 2001). To confirm that
6ß4 signaling to ERK is mediated by an SFK, cells transfected with wild-type
6ß4 were treated with the SFK inhibitor PP2. As expected,
6ß4-mediated activation of ERK was suppressed by PP2, but not by the EGF-R inhibitor AG1478. By contrast, EGF-Rmediated activation of ERK was inhibited by AG1478, but not by PP2 (Fig. 4 C). Then, we asked if localization to lipid rafts is necessary for
6ß4-mediated SFK signaling to ERK. Cells were transfected with constructs encoding
6 in combination with wild-type ß4, phosphorylation-defective ß4 (4F), palmitoylation-defective ß4 (Cys 5), or tail-less ß4 (L). Immunoblotting showed that the cells expressed comparable amounts of wild-type, palmitoylation-defective, and 4YF ß4, and somewhat higher levels of tail-less ß4 (unpublished data). Ligation of wild-type
6ß4 caused activation of ERK, whereas ligation of the palmitoylation-defective, 4YF, or tail-less mutant did not induce this event (Fig. 4 D). These results indicate that palmitoylation of ß4 and, by inference, localization of
6ß4 to lipid rafts are necessary for efficient signaling to ERK.
To address the physiological significance of 6ß4 incorporation in lipid rafts, we examined the ability of ß4 Cys 5 to promote EGF-dependent mitogenesis. After synchronization in G0, PA-JEB keratinocytes stably transduced with retroviral vectors encoding ß4, ß4 Cys 5, or empty virus were plated on laminin-5, or they were incubated with mAb 3E1 and plated on dishes coated with antimouse IgGs. BrdU incorporation and anti-BrdU staining indicated that wild-type ß4 significantly enhanced the ability of PA-JEB keratinocytes to progress through the cell cycle on laminin-5. Similar results were obtained after antibody-mediated ligation of
6ß4. By contrast, the palmitoylation-defective ß4 was not able to rescue EGF-mediated proliferation of PA-JEB keratinocytes (Fig. 4 E), providing evidence that
6ß4-dependent mitogenic signaling requires palmitoylation of ß4 and incorporation of
6ß4 in lipid rafts.
Although 6ß4, like other integrins, does not contain a kinase domain, ligation of
6ß4 causes phosphorylation of the cytoplasmic tail of ß4, and hence, recruitment of Shc and other signal transducers. In order for this to occur, the integrin must associate with a tyrosine kinase. Here, we have shown that compartmentalization in lipid rafts is necessary to couple
6ß4 to a pSFK and thus reconstitute its ability to activate signaling and promote epithelial mitogenesis. These results provide direct evidence that compartmentalization in lipid rafts is required for
6ß4 signaling. Because it is known that part of the EGF-R localizes to lipid rafts (Waugh et al., 1999), and our prior analyses have indicated that the EGF-R activates ß4 signaling through the integrin-associated pSFK (Mariotti et al., 2001), it is possible that incorporation in lipid rafts is also necessary for EGF-Rdependent activation of ß4 signaling.
How does matrix binding activate 6ß4 signaling? At steady state, only a fraction of
6ß4 is palmitoylated, and hence localized to lipid rafts. However, antibody- or ligand-induced oligomerization of
6ß4 increases the amount of integrin recovered in the raft fraction, suggesting that matrix binding to
6ß4 increases the integrin's affinity for lipid rafts. In addition, palmitoylation is a reversible process (Resh, 1999), allowing for regulated incorporation of
6ß4 in lipid rafts. We envision that matrix-induced aggregation of
6ß4-containing rafts promotes signaling by bringing the integrin in close proximity to the pSFK, and possibly by excluding a negative regulatory tyrosine phosphatase, as implied by the observation that vanadate greatly enhances phosphorylation of ß4 (Dans et al., 2001). In addition, because H-Ras, which is palmitoylated and localizes to lipid rafts, activates PI-3K more efficiently than other Ras isoforms (Yan et al., 1998; Roy et al., 1999), the association with lipid rafts may explain the ability of
6ß4 to activate PI-3K, and hence, Rac, more effectively than other integrins (Shaw et al., 1997). Thus, compartmentalization in lipid rafts potentially explains several specific aspects of
6ß4 signaling.
Although other integrins do not appear to be palmitoylated, prior reports suggest that membrane compartmentalization plays a role in signaling by many integrins. Certain ß1 and v integrins associate, through caveolin-1, with pSFKs, thereby activating Shc signaling to ERK (Wary et al., 1998). Although these integrins are soluble in Triton X-100, it is possible that they associate with lipid rafts through a Triton X-100sensitive interaction with uPAR, which is GPI linked and localized to rafts (Wei et al., 1999). The
3ß1,
6ß1, and certain other integrins associate with tetraspanins (Hemler, 2001). Because many tetraspanins are palmitoylated and also tend to form oligomers, they could promote integrin incorporation in lipid raft-like domains. Accordingly,
6ß1 associates with detergent-resistant microdomains to promote survival signaling in oligodendrocytes (Baron et al., 2003). Finally,
vß3,
IIbß3, and
2ß1 combine with the integrin-associated protein in cholesterol-dependent microdomains distinct from classical rafts (Green et al., 1999), and
4ß1 and
Lß2 have been shown to colocalize with the lipid raft marker GM-1 in T cells (Leitinger and Hogg, 2002). We anticipate that future experiments will reveal that the mechanism of membrane compartmentalization illustrated here also operates, with some variations, in other integrin-signaling systems.
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Materials and methods |
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Cells, constructs, and expression methods
Vectors encoding human 6, ß4, and the ß4 mutants B, C, E, and L were described previously (Spinardi et al., 1993; Mainiero et al., 1997). The ß4 Cys mutants were generated with QuikChange® (Stratagene). HaCat, HUVECs, rat bladder 804G, and 293-T HEK cells were transfected as described previously (Dans et al., 2001; Mariotti et al., 2001). Immortalized PA-JEB keratinocytes (Gagnoux-Palacios et al., 1997) were cultured in serum-free keratinocyte growth medium (GIBCO BRL). Retroviral particles were recovered from Phoenix packaging cells transfected with pLZRS-IRES-zeo encoding ß4 or the ß4 Cys 5 mutant, and were used to transduce PA-JEB keratinocytes.
Biochemical methods
For fractionation, cells were lysed on ice for 30 min with 0.5% Triton X-100 in TNE (25 mM Tris, pH 7.5, 150 mM NaCl, and 2 mM EDTA) with inhibitors. After Dounce homogenization, the lysates were subjected to either sucrose or OptiPrepTM gradient ultracentrifugation. When indicated, cells were pretreated with 0.2% saponin for 10 min at 4°C or with 10 mM MßCD for 1 h at 37°C before detergent extraction. To mimic ligand-induced aggregation of 6ß4, cells were detached with EDTA, incubated in suspension with 10 µg/ml anti-ß4 mAb 3E1 for 20 min on ice, washed, and then incubated with 10 µg/ml antimouse IgGs for 5 min at 37°C. For immunoblotting, proteins from each fraction were precipitated with TCA. For immunoprecipitation, the lipid raft and soluble fractions were diluted with an equal volume of TNE, 1% Triton X-100, and 10% sucrose. After labeling with Tran 35S-label (ICN Biomedicals), IC16 (Peseckis et al., 1993), or [3H]palmitate (ICN Biomedicals; Wolven et al., 1997), cells were lysed in 50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet® P-40, 0.5% sodium deoxycholate, and 2 mM EDTA with phosphatase and protease inhibitors, and were immunoprecipitated with mAb 3E1. A PhosphorImager was used to quantify the results. Kinase assays were as described previously (Mariotti et al., 2001).
Adhesion and immunofluorescence
To test adhesion, cells were plated for 1 h at 4°C or for 30 min in the presence of anti-3 on wells coated with laminin-5 matrix (Mariotti et al., 2001). The relative resistance of PA-JEB keratinocytes to trypsin/EDTA-induced detachment was measured as described previously (Gagnoux-Palacios et al., 2001). Cells were fixed with 3.7% PFA and permeabilized with 0.1% Triton X-100 before immunofluorescent staining.
Cell cycle progression
PA-JEB keratinocytes stably transduced with pLZRS, pLZRS-ß4, or pLZRS-ß4 Cys5 were deprived of growth factors, detached, and either plated on coverslips coated with 5 µg/ml human laminin-5 (CHEMICON International) or incubated in suspension with the mAb 3E1 and then plated on coverslips coated with 10 µg/ml antimouse IgGs. Cells were cultured for 16 h in serum-free keratinocyte growth medium supplemented with 10 ng/ml EGF and 50 µg/ml bovine pituitary extract, labeled with BrdU, and then stained with anti-BrdU mAbs. The results were expressed as percentage of rescue (R), according to the formula R =([X-L]/[F-L]) x 100 (where X is the percentage of BrdU-positive cells expressing ß4 Cys 5; F is the percentage of BrdU-positive cells expressing wild-type ß4; and L is the percentage of BrdU-positive cells transduced with pLZRS, on either laminin-5 or the anti-ß4 substrate).
Online supplemental materials
Fig. S1 shows a control for the coimmunoprecipitation of 6ß4 with pSFKs from the lipid raft fraction. HaCat keratinocytes were transiently transfected with a vector encoding a GPI-linked form of GFP, obtained by fusing GFP to the COOH terminus of CD55. After Triton X-100 extraction and sucrose density fractionation, the lipid raft and the soluble fractions were immunoprecipitated with anti-GFP mAbs and subjected to blotting with either anti panSrc or anti-GFP pAbs. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200305006/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health awards R37 CA58976 (to F.G. Giancotti), R01 GM57966 (to M.D. Resh), and P30 CA08748 (to the Memorial Sloan-Kettering Cancer Center).
Submitted: 6 May 2003
Accepted: 5 August 2003
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