1 Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
2 Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
*Author for correspondence (e-mail: jborst{at}nki.nl)
Accepted March 13, 2001
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SUMMARY |
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Key words: EGF receptor, Endocytosis, Cbl, Downregulation, Ubiquitin
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INTRODUCTION |
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The cellular homologue of the murine retroviral protein v-Cbl, c-Cbl (Langdon et al., 1989), is a negative regulator of a wide variety of protein tyrosine kinase-coupled membrane receptors. Three mammalian Cbl genes have been identified (Blake et al., 1991; Keane et al., 1995; Keane et al., 1999; Kim et al., 1999) and Cbl homologues have been found in Caenorhabditis elegans (Yoon et al., 1995) and Drosophila (Meisner et al., 1997; Robertson et al., 2000). The first evidence that Cbl molecules negatively regulate RTKs came from studies in C. elegans, in which loss of function of the Cbl homologue Sli-1 compensated for defective Let-23 (EGFR) signalling (Jongeward et al., 1995). Drosophila Cbl (D-Cbl) was found to suppress the development of the R7 photoreceptor neuron, which is guided by RTK signalling (Meisner et al., 1997). c-Cbl deficient mice show several morphological aberrations that indicate loss of cell growth control, such as hyperplasia of lymphoid and mammary tissues and abundant extramedullary haematopoiesis in the spleen (Murphy et al., 1998). In addition, c-Cbl-/- thymocytes express higher T cell antigen receptor levels and show enhanced ZAP-70 activity compared to wild-type cells, suggesting a defect in negative control of receptor signalling (Thien et al., 1999). In Cbl-b-/- mice, the signalling threshold for antigen-specific T cell activation is lowered and reached in the absence of CD28 signals (Bachmaier et al., 2000; Chiang et al., 2000). An explanation for the negative regulatory function of Cbl molecules was provided by the observation that c-Cbl stimulates downregulation of receptor numbers at the plasma membrane (Lee et al., 1999; Levkowitz et al., 1998; Miyake et al., 1998).
The N-terminal half of the Cbl proteins is conserved during evolution and contains a phosphotyrosine-binding region and a Ring finger domain. The phosphotyrosine-binding region is composed of a four-helix domain, an EF hand and an SH2-like domain (Meng et al., 1999), and is recruited to phosphotyrosine motifs in RTKs and the ZAP-70/Syk-family of cytoplasmic tyrosine kinases (Smit and Borst, 1997). Recently, it has been found that the Ring finger of c-Cbl interacts with ubiquitin-conjugating proteins, or E2s, and that c-Cbl is in fact a novel type of E3, or ubiquitin ligase, that is recruited to and ubiquitinates tyrosine-kinase-coupled membrane receptor systems (Joazeiro et al., 1999; Levkowitz et al., 1999; Yokouchi et al., 1999). Overexpression of c-Cbl strongly enhances ubiquitination and degradation of the EGFR, platelet-derived growth factor receptor and colony stimulating factor 1 receptor (CSF-1R) (Lee et al., 1999; Levkowitz et al., 1998; Miyake et al., 1998). c-Cbl mutants lacking a functional Ring finger domain cannot mediate receptor ubiquitination and downregulation. Such mutants include the oncogenic variants v-Cbl as well as 70Z-Cbl, which was originally isolated from the 70Z/3 mouse pre-B-lymphoma cell line and contains a 17 amino acid deletion that disrupts the Ring finger motif (Andoniou et al., 1994).
It is not clear at which point in the endocytic pathway c-Cbl comes into play, nor how it promotes the removal of receptors from the cell surface. It has been proposed that it interferes with recycling by stimulating receptor sorting to the lysosomes. In that study, c-Cbl was found to associate with the EGFR upon its arrival in endosomes (Levkowitz et al., 1998). Other investigators suggest that c-Cbl stimulates endocytosis per se. Upon CSF-1 stimulation of macrophages, c-Cbl was targeted to the plasma membrane (Wang et al., 1996) and studies on macrophages of c-Cbl-/- mice indicated that c-Cbl promotes rapid internalization of the CSF-1R (Lee et al., 1999).
We have analysed the spatiotemporal interaction between c-Cbl and the EGFR after ligand binding. We show that c-Cbl is recruited to and ubiquitinates the activated EGFR while it is still present at the cell surface. Using confocal and electron microscopy, we find c-Cbl together with the EGFR at the plasma membrane, as well as in clathrin-coated vesicles and in multivesicular bodies. Our data indicate that c-Cbl might be involved in the recruitment of the EGFR into clathrin-coated pits, as well as in sorting of the receptor to the lysosomes.
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MATERIALS AND METHODS |
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Constructs
The pMT2-HA-cCbl vector was generated by ligation of the HA-tagged c-cbl cDNA from pGEM4Z-cCbl (provided by W. Y. Langdon, University of Western Australia, Nedlands, Australia) into pMT2SM using its SalI site. The pMT123 vector containing HA-ubiquitin cDNA has been described by Treier et al. (1994). The insert was isolated using EcoRI/NotI and ligated into pMT2SM. The pMT2-dynamin-1 (wild type and K44A mutant) constructs have been described elsewhere (Kranenburg et al., 1999). The pMT2-EGFR vector was a gift of W. Moolenaar (The Netherlands Cancer Institute, Amsterdam, The Netherlands).
Transfections
COS cells (1x106 per 10 cm dish) were transfected by the DEAE-dextran method, using routinely 2.5 µg DNA per plasmid. CHO cells (3x105 per 3.5 cm well) were transfected using lipofectamine PLUS Reagent (Gibco). The following quantities of DNA were used for each construct: 3 µg of pMT2-HA-cCbl, 2 µg of pMT2-EGFR, 1 µg of pMT2-HA-ubiquitin and 4 µg of the dynamin cDNAs. The DNA mixture was added to the cells in a total volume of 1 ml of DMEM. After 4-6 hours, the DNA containing solution was removed and 3 ml medium were added. CHO cells stably expressing the human EGFR were generated by transfecting 1 µg pMT2-EGFR with 1 µg of pcDNA3 to allow selection on medium containing 2.5 mg ml-1 G418. Cells strongly expressing the EGFR were selected after 3 weeks by sorting with a MoFlo high speed sorter (Cytomation, Fort Collins, CO, USA) using anti-EGFR mAb 108.1.
Immunoprecipitation and immunoblotting
Cells were used for immunoprecipitation 48 hours after transfection. COS cells were serum starved overnight and stimulated with 25 ng ml-1 EGF (Becton Dickinson Labware, Bedford, MA, USA) on ice or at 37°C for several periods of time. After stimulation, cells were immediately put on ice, washed quickly with cold PBS and incubated for 30 minutes at 0°C with lysis buffer (1% NP-40, 30 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 mM EDTA pH 8.0, 10 mM NaF, 1 mM sodium orthovanadate, 10 µg ml-1 phenylmethylsulfonyl fluoride, 0.1 µM leupeptin and 0.1 µM aprotinin). Cell lysates were clarified by centrifugation for 10 minutes at 14,000 rpm. The appropriate antibodies were added to the lysates and incubated for 2 hours or overnight at 4°C. Immune complexes were incubated with protein A-Sepharose beads for an additional 2 hours. Precipitated proteins were subjected to SDS-PAGE and blotted to nitrocellulose. Filters were blocked for 30 minutes at room temperature or at 4°C overnight in TBST (20 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.05% Tween-20) containing 1% bovine serum albumin (BSA) or non-fat dry milk. Filters were incubated with primary antibodies for 2 hours at room temperature or overnight at 4°C, washed three times in TBST and incubated with secondary antibodies (1:7500 dilution of horseradish-peroxidase-conjugated rabbit anti-mouse Ig or swine anti-rabbit Ig) for 45 minutes at room temperature. After washing the filters in TBST, proteins were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech).
Immunofluorescence
One day after transfection, cells were detached with trypsin/EDTA and plated on coverslips placed in six-well plates. Cells were allowed to attach for 24 hours and then serum starved for 3 hours (CHO) or overnight (COS) in DMEM containing 20 mM HEPES and 0.1% BSA. Cells were stimulated with 25 ng ml-1 EGF at 37°C or on ice for the appropriate periods of time. When stimulation was performed on ice, cells were incubated for 30 minutes on ice prior to the addition of EGF. For cytosol depletion, stimulated cells were treated for 15 minutes on ice with 0.05% saponin in permeabilization buffer (80 mM PIPES pH 6.8, 0.1 mM EGTA and 0.5 mM MgCl2) prior to fixation. Cells were fixed on ice for 2 minutes with methanol kept at -20°C. Methanol was removed and coverslips were allowed to dry at room temperature. All further incubations were performed at room temperature. Cells were rehydrated in PBS for 5 minutes and nonspecific binding sites were blocked for 30 minutes using 1% BSA in PBS containing 1 mM MgCl2 and 1 mM CaCl2. Incubations were performed with antibodies diluted in blocking buffer for 45 minutes, after which coverslips were washed and incubated for 30 minutes with the appropriate FITC- or Texas-Red-conjugated secondary antibodies diluted in blocking buffer. Coverslips were washed and mounted in Vectashield (Vector Laboratories, Burlingham, CA, USA) and viewed under a Leica TCS NT confocal laser-scanning microscope (Leica Microsystems, Heidelberg, Germany). Confocal images were taken from a basal plane of the cells, just above the basal membrane, unless indicated otherwise.
Immunoelectron microscopy
COS cells plated in 10 cm petri dishes were transfected with c-cbl cDNA as described above. Cells were serum starved overnight and stimulated with 25 ng ml-1 EGF at 37°C or on ice. Cells were fixed for 24 hours in 4% paraformaldehyde in 0.1 M PHEM buffer (80 mM PIPES, 25 mM HEPES, 2 mM MgCl2 and 10 mM EGTA, pH 6.9) and processed for ultrathin cryosectioning as described (Calafat et al., 1997). 45-nm cryosections were cut at -125°C using diamond knives (Drukker Cuijk, The Netherlands) in an ultracryomicrotome (Leica Aktiengesellschaft, Vienna, Austria) and transferred with a mixture of sucrose and cellulose onto formvar-coated copper grids (Liou et al., 1996). The grids were placed on 35-mm petri dishes containing 2% gelatin. For single immunolabelling, the sections were incubated with antibodies for 45 minutes, followed by 30 minutes incubation with 10-nm protein-A-conjugated colloidal gold (Department of Cell Biology, Utrecht University Medical Center, Utrecht, The Netherlands). For double immunolabelling, the procedure described by Slot et al. (1991) was followed with 10-nm and 15-nm protein-A-conjugated colloidal gold probes. As control, primary antibody was replaced by irrelevant rabbit antiserum. After immunolabelling, the cryosections were embedded in a mixture of methylcellulose and uranyl acetate and examined with a Philips CM 10 electron microscope (Eindhoven, The Netherlands).
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RESULTS |
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DISCUSSION |
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Levkovitz et al. (1998), however, described c-Cbl as a resident protein of endosomes, which enhances EGFR downregulation by promoting sorting of the receptor to lysosomes. They demonstrated that, under conditions of v-Cbl overexpression, the EGFR was rescued from sorting to lysosomes and recycled back to the cell surface. v-Cbl did not affect localization of the EGFR in endosomes or decrease the rate of ligand uptake. Therefore, they did not consider the possibility that c-Cbl might play a role in receptor internalization. The discrepancy with our data can be resolved, because Levkovitz et al. used high concentrations of EGF, which might saturate the clathrin-dependent internalization pathway (Wiley, 1988). Under such conditions, EGFR use a ligand- and clathrin-independent internalization pathway (Sorkin, 1998; Watts and Marsh, 1992), which is presumably not regulated by c-Cbl.
Possibly, c-Cbl provides an internalization signal to the ligand-receptor complex. Signals that mediate constitutive binding to clathrin-coated pits include tyrosine-based motifs and the dileucine motif (Sorkin, 1998). The motif YXX (X is any amino acid,
has a bulky hydrophobic group), which is present in, for instance, the transferrin receptor, provides a binding site for the adaptor protein AP-2, a heterotetrameric protein that induces the assembly of clathrin triskelions at the plasma membrane. The EGFR can interact directly with AP-2 via the tyrosine-based motif FYRAL (Sorkin et al., 1996). Whereas transferrin receptor internalization was found to be dependent on the residues in AP-2 that interact with the tyrosine motif, ligand-induced EGFR endocytosis was not (Nesterov et al., 1999). Therefore, clathrin-mediated internalization of the EGFR must depend on another signal. We suggest that c-Cbl-mediated ubiquitination of the EGFR provides such a signal. We have demonstrated that c-Cbl ubiquitinates the EGFR prior to its internalization. In agreement with our findings, the EGFR was recently found to be ubiquitinated in Hela cells overexpressing K44A dynamin (Stang et al., 2000).
There is ample evidence from the yeast system that ubiquitination serves a role in receptor endocytosis. A chimeric receptor consisting of a single ubiquitin fused to a cytoplasmic tail-truncated Ste2p receptor was effectively internalized. Apparently, ubiquitin sufficed as an internalization signal in this case (Shih et al., 2000). Whereas monoubiquitination is sufficient (Terrell et al., 1998), multiple ubiquitination increased the internalization rate of the Ste3p receptor (Roth and Davis, 2000). In case of the yeast uracil permease, lysine 63-linked diubiquitination was needed to obtain maximal internalization rates (Galan and Haguenauer-Tsapis, 1997). Similar to the findings in yeast, a single ubiquitin molecule fused to the cytoplasmic region of the invariant chain or the interleukin-2 receptor chain was sufficient to mediate receptor internalization in Hela cells (Nakatsu et al., 2000). Moreover, plasma membrane levels of an ubiquitination-defective mutant of the epithelial sodium channel (ENaC) were increased (Staub et al., 1997). In case of the human growth hormone receptor, interaction with components of the ubiquitin-conjugating machinery is required for its endocytosis, whereas receptor ubiquitination does not seem to be involved (Govers et al., 1999; Strous et al., 1996).
Several other molecules have been implicated in recruitment of the EGFR into clathrin-coated pits, such as Grb-2, Eps15 and c-Src. The Grb-2 adaptor protein can bind with its SH2 domain to the cytoplasmic tail of the EGFR, whereas its SH3 domains can interact with dynamin (Seedorf et al., 1994; Wang and Moran, 1996). Overexpression of the Grb-2 SH2 domain was found to inhibit EGFR internalization (Wang and Moran, 1996). Eps15 and Eps15R are substrates of the activated EGFR and are essential for receptor internalization (Carbone et al., 1997). Tyrosine phosphorylation of Eps15 is required for ligand-induced but not for constitutive endocytosis (Confalonieri et al., 2000). Eps15 interacts with AP-2 and clathrin but does not bind directly to the EGFR, implying the involvement of other components (van Delft et al., 1997). The protein tyrosine kinase c-Src, which is activated by the EGFR, has been implicated in the exit of the activated EGFR from caveolae, as well as its internalization via clathrin-coated pits (Carpenter, 2000; Ware et al., 1997). c-Src phosphorylates the clathrin heavy chain and might thus control the assembly of a clathrin network (Wilde et al., 1999).
c-Cbl can easily be visualized as intermediate in Grb-2- and Src-regulated receptor endocytosis, because both molecules can interact via their SH3 domains with the proline-rich region in the C-terminus of c-Cbl. Furthermore, phosphorylated tyrosine residues in the C-terminus serve as docking sites for the SH2 domains of Vav, p85 and the adaptor protein Crk (Lupher et al., 1999; Smit and Borst, 1997; Thien and Langdon, 1998). It has been hypothesized that c-Cbl interacts with Eps15 via Crk (Smit and Borst, 1997), thus providing the link between Eps15 and the EGFR. Together, these data suggest that the C-terminal region of c-Cbl provides additional cues for receptor internalization.
Our biochemical and confocal data showed a prolonged association of c-Cbl with the activated EGFR. Immunogold electron microscopy allowed detection of c-Cbl in the internal vesicles of multivesicular bodies. We conclude that the c-Cbl-EGFR interaction is maintained throughout the endocytic pathway. According to the model of Futter et al. (1996), recycling receptors are lost from maturing multivesicular bodies, whereas the EGFR becomes localized in internal vesicles and ends up in lysosomes upon fusion of both organelles. This suggests that c-Cbl will be degraded together with the EGFR in lysosomes. We find a small decrease in c-Cbl levels in total lysates of stimulated COS cells in time (Fig. 1). Others have found no evidence for degradation of c-Cbl upon activation of the CSF-1R and describe that c-Cbl is relocated to the cytosol after receptor internalization (Wang et al., 1996). These differences might be receptor specific.
We propose a model in which c-Cbl-mediated ubiquitination facilitates recruitment of activated EGFRs into clathrin-coated pits by providing a ligand-dependent internalization signal. In this way, c-Cbl enhances internalization of the ligand-receptor complex via the saturatable, clathrin-mediated endocytosis pathway. Subsequently, c-Cbl might be involved in receptor sorting and finally is routed, together with the EGFR, towards the lysosomes.
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ACKNOWLEDGMENTS |
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