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The Endocytosis-Linked Protein Dynamin Associates with Caveolin-1 and Is Tyrosine Phosphorylated in Response to the Activation of a Noninternalizing Epidermal Growth Factor Receptor Mutant

Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706-1532

Address all correspondence and requests for reprints to: Paul J. Bertics, Ph.D., Department of Biomolecular Chemistry, University of Wisconsin, 1300 University Avenue, Madison, Wisconsin 53706-1532. E-mail: . pbertics{at}facstaff.wisc.edu

	Abstract

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Several studies have shown that an EGF receptor C-terminal truncation at residue 973 (CT973) attenuates ligand-induced receptor endocytosis and is associated with cell transformation. Previously, we have shown that EGF stimulation of murine B82L fibroblasts expressing CT973 EGF receptors can promote the tyrosine phosphorylation of caveolin-1, which is a major component of caveolae membranes. Because dynamin plays an essential role in receptor-mediated endocytosis via clathrin-coated pits and caveolae, and because dynamin has been localized to caveolae, we tested the hypothesis that dynamin associates with caveolin-1 and is differentially modified in response to the abnormal actions of internalization-defective EGF receptors. We found that dynamin coimmunoprecipitates with caveolin-1 in cells containing normal or CT973 EGF receptors, but EGF stimulated the tyrosine phosphorylation of dynamin only in cells expressing truncated/oncogenic EGF receptors. Maximum dynamin phosphorylation was observed within 15 min of EGF administration and decreased thereafter. Furthermore, phosphotyrosine-containing proteins in the dynamin immunocomplexes were observed to be reactive with anticaveolin-1 antibodies. The EGF receptor does not appear to directly phosphorylate dynamin because a Src antagonist, PP1, inhibited the EGF-induced tyrosine phosphorylation of dynamin at a concentration that does not block EGF receptor autophosphorylation. These results provide the first evidence that caveolin-1 and dynamin form a complex, and that the EGF-induced tyrosine phosphorylation of dynamin occurs via a Src inhibitor-sensitive signaling pathway that is associated with the aberrant actions induced by internalization-defective EGF receptors.

	Introduction

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EPIDERMAL GROWTH FACTOR (EGF) is a small polypeptide growth factor that exerts a wide variety of biological effects, including the stimulation of cell proliferation, migration, and differentiation (1, 2). The receptor for EGF is a transmembrane glycoprotein that consists of an extracellular EGF binding domain at its amino terminus, a transmembrane spanning region, a cytoplasmic EGF-stimulable protein-tyrosine kinase domain, and a regulatory carboxy (C)-terminal domain that contains multiple autophosphorylation sites (1). Multiple studies have shown that the C terminus of the EGF receptor can regulate receptor kinase activity, substrate specificity, and trafficking (1, 2). Consequently, truncation of the C terminus of the receptor at residue 973 (CT973), which deletes all autophosphorylation sites and abolishes ligand-induced down-regulation of the receptor, allows for an enhanced and/or altered EGF-stimulated tyrosine phosphorylation of protein substrates in vivo (3) and promotes cell transformation (4, 5). These studies suggest that truncated EGF receptors have an altered specificity for target proteins, and that they may establish unique signaling pathways in addition to those associated with normal EGF receptor function. In this regard, we have shown that the activation of truncated/oncogenic EGF receptors can enhance the tyrosine phosphorylation of caveolin-1, which is a major component of caveolae membrane (6). Subsequent to ligand binding, the EGF receptor is internalized through clathrin-coated pits, and this process has been suggested to modulate receptor signal transduction (7, 8).

Recent studies have also shown that many cell surface receptors and intracellular signaling molecules, including the EGF receptor and Src family kinases, are concentrated in specialized plasma membrane domains known as caveolae (9, 10). These caveolae are reported to be sites where certain EGF-induced signaling events are initiated (9). Under normal circumstances, EGF stimulation results in a down-regulation of the total level of cellular EGF receptors, including a drop in the percentage of EGF receptors localized to caveolae, but oncogenic EGF receptors, such as the internalization- impaired CT973 receptor, fail to move out of caveolae and do not down-regulate following EGF stimulation (11). These studies suggest that, following ligand exposure, poorly internalizing forms of the EGF receptor may reside for excessive periods of time in caveolae after EGF binding, thereby inducing unique or chronic signaling events that can be deleterious to the cell.

The GTPase dynamin mediates the internalization of both clathrin-coated pits and caveolae through a GTP-dependent fission process, and recent studies have shown that dynamin is localized to clathrin-coated pits and caveolae (10, 12, 13, 14). Expression of a dominant-negative form of dynamin (K44A), which lacks GTPase activity, blocks EGF-induced receptor down-regulation and increases EGF-dependent mitogenesis (15). In addition, dynamin has been reported to interact with the adapter protein Grb2, which associates with the activated EGF receptor, and this process facilitates EGF receptor internalization (16). Given that dynamin is localized to caveolae (10), and that a poorly internalizing/oncogenic EGF receptor (CT973) constitutively resides in caveolae (11), the present study focused on examining dynamin interaction with the major caveolae structural protein, caveolin-1, and evaluated the possible participation of dynamin in the EGF-induced signal transduction events initiated by oncogenic EGF receptors. Indeed, although these mutant EGF receptors are known to traffic more slowly (11) and to induce altered patterns of intracellular protein-tyrosine phosphorylation (3, 4, 5, 6), very little is known concerning specific proteins in cellular trafficking that are differentially modified in response to EGF receptor mutation. Accordingly, the present study provides the first insight into how internalization-defective/transforming EGF receptors influence the interaction and phosphorylation of several key proteins recently linked to normal EGF receptor trafficking (8, 11, 12).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Materials
Murine B82L cells expressing wild-type (WT) or truncated (CT973) human EGF receptors were generated as described (6). DMEM was purchased from Life Technologies, Inc. (Grand Island, NY), and cosmic calf serum was obtained from HyClone Laboratories, Inc. (Logan, UT). Human EGF and an anti-Src antibody (GD11, which was used for immunoblotting) were acquired from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal anti-Src family kinase antibodies (sc-18, which were used for immunoprecipitation), polyclonal anticaveolin-1 antibodies (sc-894), protein A-agarose, horseradish peroxidase-conjugated goat antimouse IgG and goat antirabbit IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antiphosphotyrosine antibodies 4G10 and PY-20 were obtained from Upstate Biotechnology, Inc. and Transduction Laboratories, Inc. (Lexington, KY), respectively. Monoclonal antiphospho-caveolin-1 antibodies, which react only with the forms of caveolin-1 that are phosphorylated on tyrosine-14, were obtained from Transduction Laboratories, Inc. Monoclonal antiactive ERK1 and ERK2 antibodies, which react only with the activated forms of ERK1/ERK2 that are dually phosphorylated on threonine and tyrosine, were purchased from Promega Corp. (Madison, WI). Polyclonal antidynamin antibodies (-dyn 61, -dyn 63, and -dyn 65) were a gift of Dr. Mark McNiven (Mayo Clinic, Rochester, MN). Immobilon-P polyvinylidene difluoride membranes (0.45 µm) were acquired from Millipore Corp. (Bedford, MA). Micro BCA protein assay reagents were purchased from Pierce Chemical Co. (Rockford, IL). Chemiluminescent reagents were bought from Kirkegaard \|[amp ]\| Perry Laboratories (Gaithersburg, MD). The Src kinase inhibitor PP1 was obtained from BIOMOL (Plymouth Meeting, PA). All other reagents were purchased from Sigma (St. Louis, MO).

Cell culture
Murine B82L fibroblasts that were transfected with WT EGF receptor (B82L-WT cells) or with the CT973 EGF receptor (B82L-CT973 cells) were cultured in DMEM containing 10% cosmic calf serum and 10 µM methotrexate (which was added because a mutant dihydrofolate reductase gene was used as a selectable marker for CT973 EGF receptor expression) (17). The cells were grown for 3 d to approximately 70% confluence and were serum-starved overnight using 0.1% BSA in DMEM before treatment. In addition, these cells have been previously shown to express comparable levels of WT and CT973 EGF receptors using flow cytometry (6).

Immunoprecipitation
Serum-starved cells were treated with 50 nM EGF or vehicle (20 mM HEPES, pH 7.4), and then lysed in an immunoprecipitation assay (IPA) buffer [10 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.05% SDS, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml leupeptin and 1 µg/ml aprotinin]. The cell lysates were centrifuged at 10,000 x g for 10 min at 4 C and the supernatants were precleared by incubating with protein A-agarose beads for 1 h at 4 C, followed by centrifugation at 10,000 x g for 1 min at 4 C. The supernatants were subjected to immunoprecipitation using 2 µg of either an anticaveolin-1, anti-Src, or antidynamin antibody; nonspecific rabbit IgG was used as a control. The samples were incubated overnight followed by incubation with protein A-agarose beads for 2 h at 4 C. After washing the beads three times with ice-cold IPA buffer containing detergent and three times with IPA buffer without detergent, the bound proteins were eluted by the addition of 2x SDS-PAGE sample buffer [2% SDS, 20 mM dithiothreitol, 1 mM Na₃VO₄, 2 mM EDTA, 10% glycerol, 20 mM Tris (pH 8.0)] followed by electrophoresis and immunoblot analysis.

Preparation of cell lysates and immunoblotting
Murine B82L cells were lysed in 1 ml of 2x SDS-PAGE sample buffer and the protein level of each sample was determined by the Micro BCA assay (Pierce Chemical Co., Rockford, IL). Equal amounts of protein (30–50 µg) were separated by SDS-PAGE using 10% gels followed by transfer to polyvinylidene difluoride membranes (6). The membranes were blocked in a TBST buffer [10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween-20] containing 0.25% gelatin at 4 C and then incubated for 1 h at 37 C with antiphosphotyrosine antibodies (2 µg 4G10 and 2 µg PY-20 in 5 ml of 0.25% gelatin/TBST). For immunoblotting analysis using other antibodies, the membranes were blocked at 4 C with 5% nonfat dry milk in TBST (milk/TBST), and then incubated with 2 µg of the primary antibody in milk/TBST for 1 h at 37 C. Next, the membranes were washed three times in TBST, incubated with horseradish peroxidase-conjugated goat antimouse IgG or goat antirabbit IgG secondary antibodies for 1 h at 37 C, and then washed three times with TBST. The labeled proteins were visualized by the enhanced chemiluminescence method. For subsequent probing of the same membrane for other proteins, the membrane was incubated in a stripping buffer (62.5 mM Tris, pH 6.8; 2% SDS; 100 mM dithiothreitol) at 70 C for 30 min, washed extensively, reblocked with milk/TBST and then reprobed with the specified antibodies as outlined above.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Coimmunoprecipitation of dynamin and caveolin-1
Dynamin has been localized to caveolae and it has been reported to be required for the internalization of caveolae membranes (10, 12). In addition, antidynamin antibodies have been used to immunoisolate caveolae membranes (13, 14), although it is not known if dynamin can interact with the major structural protein of caveolae, caveolin-1, or if their colocalization is dependent on an intact membrane. Thus, to test whether dynamin forms a stable complex with caveolin-1, we solubilized B82L cell membranes using IPA buffer containing 1% Triton X-100, 0.5% deoxycholate, and 0.05% SDS, followed by the immunoprecipitation of dynamin. These dynamin immunoprecipitates were then examined for the presence of caveolin-1 via immunoblotting analyses. As shown in Fig. 1A, caveolin-1 was observed to coimmunoprecipitate with dynamin when using solubilized extracts from B82L fibroblasts expressing comparable levels of cell surface WT or truncated CT973 EGF receptors. In addition, it was noted that similar levels of dynamin were detected in B82L cells expressing either the WT or CT973 EGF receptor (Fig. 1B), and comparable amounts of dynamin could be immunoprecipitated from extracts of both cell types (Fig. 1A). Furthermore, nonspecific antibodies did not precipitate dynamin or caveolin-1, and the coimmunoprecipitation of caveolin-1 with dynamin was not strictly EGF dependent, i.e. this interaction was readily detected in cells grown under serum-free conditions. However, a slower migrating band (24 kDa) that was immunoreactive with anticaveolin-1 antibodies was observed in the dynamin immunoprecipitates prepared from the lysates of EGF-stimulated cells expressing the CT973 EGF receptor (Fig. 1A). This protein appears to be a modified form of caveolin-1 as described previously (6). These results reveal that dynamin either directly or indirectly forms a complex with caveolin-1, and that this interaction is detergent resistant. Moreover, the continual association of a pool of dynamin and caveolin-1 may allow for more efficient communication between caveolae/caveolin-1 and dynamin-dependent processes such as receptor trafficking (8, 11, 12).

View larger version (45K): [in this window] [in a new window]   Figure 1. Coimmunoprecipitation of dynamin with caveolin-1. A, Serum-starved B82L-WT and CT973 cells were stimulated without (-) or with 50 nM EGF (+) and total cell lysates were subjected to immunoprecipitation (IP) using antidynamin antibodies or nonspecific IgG (NS) as a control for immunoprecipitation. Immunoprecipitates were analyzed by immunoblotting (IB) using either anti-dynamin antibodies (-dyn 61) or anticaveolin-1 (-cav) antibodies. Similar results were also observed in two independent experiments. B, Aliquots of total cell protein (30 µg) from B82L-WT and B82L-CT973 cells were subjected to immunoblot analysis using either anti-dynamin or anti-ERK1 and ERK2 (-ERK1&2) as a loading control. Similar results were also observed in two independent experiments.

View larger version (83K): [in this window] [in a new window]   Figure 2. EGF-stimulated tyrosine phosphorylation of dynamin and caveolin-1-associated proteins. Serum-starved B82L-WT and B82L-CT973 cells were stimulated without (-) or with 50 nM EGF (+) and 500 µg of cell lysates were subjected to immunoprecipitation using either anticaveolin-1 (-cav) or antidynamin (-dyn 61) antibodies. Immunoprecipitates were analyzed by immunoblotting using either antiphosphotyrosine (-pY), antidynamin (-dyn) or anticaveolin-1 (-cav) antibodies. Similar results were observed in two independent experiments. The upper right-hand panel represents a longer exposure of the CT973/-cav lanes shown in the upper left-hand panel.

View larger version (32K): [in this window] [in a new window]   Figure 3. Tyrosine phosphorylation of dynamin immunoprecipitated from EGF-treated cells expressing a truncated CT973 EGF receptor. Serum-starved B82L-WT and B82L-CT973 cells were stimulated without (-) or with (+) 50 nM EGF, and cell lysates (500 µg of cell proteins) were subjected to immunoprecipitation using two different antidynamin antibodies (-dyn 63 and -dyn 65). The immunoprecipitates were analyzed by immunoblotting using either antiphosphotyrosine (-pY) antibodies or antidynamin (-dyn 61) antibodies. Similar results were observed in two independent experiments.

View larger version (47K): [in this window] [in a new window]   Figure 4. Time course of EGF-induced tyrosine phosphorylation of dynamin. Serum-starved B82L-CT973 cells were treated with 50 nM EGF for 0–60 min, and the cell lysates were subjected to immunoprecipitation using antidynamin (-dyn 61) antibodies. Immunoprecipitates were subjected to immunoblot analysis using antiphosphotyrosine (-pY), antidynamin (-dyn 61) or anticaveolin-1 (-cav) antibodies. These experiments were performed two separate times with comparable results.

Src kinases and EGF-stimulated tyrosine phosphorylation of dynamin
The Src family of tyrosine kinases have been implicated in caveolin-1 phosphorylation (6) and in dynamin phosphorylation upon ß2-adrenergic receptor activation (18). Because Src has been found to associate with dynamin in HEK-293 cells (18), PC12 cells (20) and in B82L fibroblasts (data not shown), and because dynamin coprecipitates with caveolin-1 (Fig. 1), it is possible that Src family kinases are involved in EGF-stimulated dynamin phosphorylation. Accordingly, we evaluated the phosphorylation status of Src family kinases in B82L cells expressing either the WT or CT973 EGF receptor. The Src family kinases (Src, Fyn, Yes) were first immunoprecipitated from B82L cell lysates, and the precipitates were then immunoblotted using antiphosphotyrosine antibodies. As shown in Fig. 5A, EGF stimulation increased the tyrosine phosphorylation of Src kinases in B82L cells expressing either the WT or the CT973 EGF receptor, but the level of tyrosine phosphorylation was enhanced in cells possessing the CT973 EGF receptor. This apparent increase in phosphorylation does not appear to be due to a greater recovery of Src from EGF-stimulated B82L-CT973 cells (Fig. 5B). On the contrary, less Src was immunodetectable in the immunoprecipitates obtained from EGF-stimulated B82L-CT973 cells (Fig. 5A). Because comparable levels of Src are observed in immunoblots of total cell extracts (Fig. 5B), the loss of immunodetectable Src in the immunoprecipitates is likely a consequence of a modification (e.g. tyrosine phosphorylation) that leads to diminished reactivity of Src with the antibody used for immunoprecipitation (as observed previously, see Ref. 6), rather than a large-scale EGF-induced degradation of Src. In fact, treatment of the cells with a Src family kinase inhibitor (Fig. 5C), reverses this diminished recovery of Src in these immunoprecipitates, suggesting that phosphorylation is a factor contributing to this effect. To summarize, even though less Src appears to be immunoprecipitated from EGF-treated B82L-CT973 cells (Fig. 5, A and C), the limited material that is isolated appears heavily phosphorylated, suggesting that the mutant receptor is a powerful regulator of Src family kinases.

View larger version (39K): [in this window] [in a new window]   Figure 5. EGF-stimulated phosphorylation of Src in B82L cells. A, Serum-starved B82L-WT and B82L-CT973 cells were treated with 50 nM EGF for 5 min, and cell lysates were subjected to immunoprecipitation using an anti-Src antibody (-Src, sc-18). The immunoprecipitates were subjected to immunoblot analysis using either antiphosphotyrosine (-pY) or anti-Src (GD11) antibodies. These experiments were performed two separate times with comparable results. B, Serum-starved B82L-WT and B82L-CT973 cells were treated with 50 nM EGF for 1, 5, and 10 min. Aliquots of total cell protein (30 µg) were subjected to immunoblot analysis using anti-Src (GD11) antibodies or were blotted with anti-ERK1 and -ERK2 (-ERK1&2) antibodies as a loading control. These experiments were performed two separate times with comparable results. C, Serum-starved B82L-CT973 cells were pretreated with the Src family kinase antagonist, PP1 (3 µM and 10 µM), for 15 min and then treated with 50 nM EGF for 5 min. The cell lysates were subjected to immunoprecipitation using anti-Src antibodies (-Src, sc-18), followed by immunoblot analysis using either antiphosphotyrosine antibodies (-pY) or anti-Src antibodies (-Src, GD11).

View larger version (29K): [in this window] [in a new window]   Figure 6. Influence of a Src family kinase inhibitor on the EGF-induced tyrosine phosphorylation of dynamin, caveolin-1 and total cell protein in B82L-WT and B82L-CT973 cells. A, Serum-starved B82L-CT973 cells were pretreated with the Src antagonist, PP1 (3 µM), for 15 min and then treated with 50 nM EGF for 5 min. The cell lysates were subjected to immunoprecipitation using antidynamin antibodies (-dyn 61). The immunoprecipitates were subjected to immunoblot analysis using antiphosphotyrosine (-pY), antidynamin, or anticaveolin-1 (-cav) antibodies. These experiments were performed two separate times with comparable results. B and C, Serum-starved B82L-CT973 or B82L-WT cells were pretreated without and with the Src antagonist PP1 (3 µM) or PP2 (3 µM) for 15 min, and the cells were then treated with 50 nM EGF for 5 min. Aliquots (30 µg) of total cell protein were subjected to immunoblot analysis using antiphosphotyrosine, antiactive ERK1 and ERK2 (-active ERK1&2), antiphospho-caveolin-1 (-phospho Cav), and anti-ERK1 and -ERK2 (-ERK1&2) antibodies as a loading control. These experiments were performed two separate times with comparable results.

	Acknowledgments

 
The authors would also like to thank Dr. Mark A. McNiven (Mayo Clinic, Rochester, MN) for his generous gift of antidynamin antibodies.

	Footnotes

 
This work was funded by NIH Grant R01-GM-53271 (to P.J.B.).

Abbreviations: C, Carboxy; EGF, epidermal growth factor; WT, wild-type.

Received October 2, 2001.

Accepted for publication January 30, 2002.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, Y.-N. Articles by Bertics, P. J. Search for Related Content PubMed PubMed Citation Articles by Kim, Y.-N. Articles by Bertics, P. J.