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Constitutive and Growth Factor-Regulated Phosphorylation of Caveolin-1 Occurs at the Same Site (Tyr-14) in Vivo: Identification of a c-Src/Cav-1/Grb7 Signaling Cassette

Department of Molecular Pharmacology and The Albert Einstein Cancer Center (H.L., D.V., F.G., M.P.L.) Department of Cell Biology and The Albert Einstein Cancer Center (P.I., P.E.S.) Department of Pathology and The Albert Einstein Cancer Center (D.B.B.) Departments of Developmental & Molecular Biology (DMB) and Medicine; and the Albert Einstein Cancer Center (B.B., R.G.P.) Albert Einstein College of Medicine Bronx, New York 10461
Department of Pathology (D.M.L.) Washington University School of Medicine St. Louis, Missouri 63110
BD Transduction Laboratories (M.T.W., R.C.-G.) Lexington, Kentucky 40511

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Caveolin-1 was first identified as a phospho-protein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Caveolae are small omega-shaped indentations of the plasma membrane that have been implicated in signal transduction and vesicular transport processes (1, 2, 3, 4). Caveolae are found in most cell types but are extremely abundant in terminally differentiated cell types: adipocytes (5, 6, 7), simple squamous epithelia (type I pneumocytes and endothelial cells) (8), smooth muscle cells (9), and fibroblasts (10).

Caveolin, a 21- to 24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo (11, 12, 13, 14, 15). Recent studies have shown that caveolin is only the first member of a growing gene family of caveolin proteins; caveolin has been retermed caveolin-1. Three different caveolin genes (Cav-1, Cav-2, and Cav-3) encoding four different subtypes of caveolin have been described thus far (2, 3, 4, 16). There are two subtypes of caveolin-1 (Cav-1 and Cav-1ß) that differ in their respective translation initiation sites (17). The tissue distribution of caveolin-2 mRNA and protein is extremely similar to caveolin-1 (7, 18). In striking contrast, caveolin-3 mRNA and protein are expressed mainly in muscle tissue types (skeletal, cardiac, and smooth) (16, 19, 20).

Several independent lines of evidence suggest that caveolins may act as scaffolding proteins within caveolae membranes. In support of this assertion, 1) both the N-terminal and C-terminal domains of caveolin-1 face the cytoplasm (17, 21, 22, 23, 24); 2) caveolins exist within caveolae membranes as high molecular mass oligomers (18, 20, 23, 25, 26); 3) these caveolin oligomers have the capacity to self-associate in vitro to form larger structures that resemble caveolae (23); 4) caveolins copurify with cytoplasmic lipid-modified signaling molecules including heterotrimeric G proteins, Src family tyrosine kinases, and Ras-related GTPases (27, 28, 29, 30, 31); and 5) recombinant caveolin-1 interacts directly with heterotrimeric G proteins (32), c-Src (33) and H-Ras (30). Thus, we and others have proposed the "caveolae signaling hypothesis," which states that caveolins may serve as oligomeric docking sites for organizing and concentrating signaling molecules within caveolae membranes (1, 2, 3, 4, 23).

It has been suggested that fatty acylation may represent a common mechanism for targeting cytoplasmic signaling molecules to caveolae (1, 29, 34). In direct support of this hypothesis, many proteins (G protein -subunits, Src-family tyrosine kinases, Ras-related GTPases, and endothelial nitric oxide synthase) that copurify with caveolins undergo myristoylation, palmitoylation, prenylation, or dual acylation (1, 28, 35). These results indirectly suggest that acylation of lipid-modified signaling molecules may be required for their caveolar targeting in vivo.

Caveolins may facilitate this lipid-based targeting as: 1) caveolin-1 requires cholesterol for insertion into model lipid membranes (36, 37); 2) the membrane-spanning domain of caveolin-1 has a specific and high-affinity for fatty acyl moieties (38, 39); and 3) the C-terminal domain of caveolin-1 undergoes palmitoylation on three cysteines (residues 133, 143, and 156) (22). However, palmitoylation of caveolin-1 is not required for its membrane attachment or targeting to caveolae (22), suggesting that this modification may serve another role—perhaps in trapping other acylated proteins in the vicinity of caveolin molecules.

Historically, caveolin-1 was first identified as a major tyrosine phosphorylated protein in v-Src-transformed chicken embryo fibroblasts (40). Based on this observation, Glenney and co-workers (40) have proposed that caveolin-1 may represent a critical target during cellular transformation. The functional consequences of the phosphorylation of caveolin-1 on tyrosine are not yet known. At steady state, caveolin-1 is not phosphorylated on tyrosine (6). This is in contrast to v-Src-transformed cells where caveolin-1 is constitutively phosphorylated on tyrosine (11). However, caveolin tyrosine phosphorylation also occurs in normal cells but in a tightly regulated fashion (41). If 3T3-L1 adipocytes are serum starved and rapidly stimulated with insulin, caveolin transiently undergoes phosphorylation on tyrosine (41). It has been suggested that insulin-stimulated tyrosine phosphorylation of caveolin occurs indirectly via an endogenous Src-family tyrosine kinase, rather than directly via the insulin receptor. In support of this idea, caveolin-1 can be purified as part of a hetero-oligomeric complex that contains c-Src and other Src-family tyrosine kinases (27, 28, 30, 33).

Recently, we began to study the phosphorylation of caveolin-1 by Src family tyrosine kinases both in vitro and in vivo (42). Using purified recombinant components, we first reconstituted the phosphorylation of caveolin-1 by Src kinase in vitro. Microsequencing of Src-phosphorylated caveolin-1 revealed that phosphorylation occurs within the extreme N-terminal region of full-length caveolin-1, between residues 6–26. This region contains three tyrosine residues at positions 6, 14, and 25. Deletion mutagenesis demonstrated that caveolin-1 residues 1–21 are sufficient to support this phosphorylation event, implicating tyrosine 6 and/or 14. In vitro phosphorylation of caveolin-1-derived synthetic peptides and site-directed mutagenesis directly showed that tyrosine 14 is the principal substrate for Src kinase (42). In support of these observations, tyrosine 14 is the only tyrosine residue within caveolin-1 that bears any resemblance to the known recognition motifs for tyrosine kinases (KYVDSEGHLpY; [RK] - x(2, 3, 4) - [DE] - x(2, 3) - pY). To confirm or refute the in vivo relevance of these in vitro studies, we analyzed the tyrosine phosphorylation of endogenous caveolin-1 in v-Src-transformed NIH 3T3 cells. In vivo, two isoforms of caveolin-1 are known to exist: Cav-1 contains residues 1–178 and Cav-1ß contains residues 32–178. Only Cav-1 underwent tyrosine phosphorylation in v-Src-transformed NIH 3T3 cells, although Cav-1ß is well expressed in these cells. As Cav-1ß lacks residues 1–31 (and therefore tyrosine 14), these in vivo studies indirectly demonstrated the validity of our in vitro studies (42).

Here, we directly examine the phosphorylation of caveolin-1 on tyrosine 14 in vivo. For this purpose, we generated and extensively characterized a novel phospho-specific monoclonal antibody (mAb) probe that selectively recognizes only tyrosine 14-phosphorylated caveolin-1. Our results provide in vivo support for the hypothesis that certain tyrosine-kinase-mediated transmembrane signaling events are initiated at caveolae membranes and that caveolin-1 may function as a signaling molecule.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Characterization of a mAb Probe Specific for Tyrosine 14-Phosphorylated Caveolin-1
We previously showed that tyrosine 14 is the principal site of caveolin-1 phosphorylation by c-Src in vitro (42). To detect this phosphorylation event in vivo, we have generated a novel mAb probe that recognizes only caveolin-1 phosphorylated on tyrosine 14 (see Materials and Methods). A tyrosine-phosphorylated caveolin-1 peptide [SEGHL(pY)TVPI, residues 9–18] was used to immunize mice. Three positive clones were obtained (cl 34A, 49, and 56). Clone 56 gave the strongest immunoreactivity by Western blot analysis and was selected for detailed characterization.

Figure 1 shows the selectivity of antiphosphocaveolin-1 IgG. A glutathione-S-transferase (GST)-fusion protein carrying the N-terminal domain of caveolin-1 (residues 1–101) was purified from normal bacteria (BL21) or from a bacterial strain that harbors a tyrosine kinase (TKB1). Note that clone 56 recognized only tyrosine-phosphorylated caveolin-1 produced in the TKB1 strain, despite equal protein loading (Fig. 1A). We also reconstituted this tyrosine phosphorylation event in vivo. Cos-7 cells were transiently transfected with caveolin-1 and c-Src, alone or in combination. Figure 1B shows that antiphosphocaveolin-1 IgG only recognizes caveolin-1 when it is coexpressed with c-Src. Tyrosine-phosphorylated caveolin-1 migrated as multiple bands, with a major band at approximately 24–28 kDa. Normally, nonphosphorylated caveolin-1 migrates at approximately 22–24 kDa. In addition, we find that when tyrosine 14 is mutated to alanine (Y14A), preventing phosphorylation at this site, this immunoreactivity is completely abolished (Fig. 1B). We further tested the specificity of antiphosphocaveolin-1 IgG by peptide competition with caveolin-1-derived peptides (see Table 1 and Fig. 1C). Immunoreactivity was abolished by preincubation with a 100-fold molar excess of the antigenic peptide (PY14). Importantly, no inhibitory effect was observed with the nonphosphorylated version of the same peptide (Y14) or two irrelevant phosphopeptides (PY100 and PY148). In addition, when caveolins-1, -2, and -3 were cotransfected with activated c-Src (Y529F), only caveolin-1 was detectable with antiphosphocaveolin-1 IgG (data not shown). These results demonstrate the high selectivity of this novel mAb probe for tyrosine 14-phosphorylated caveolin-1.

View larger version (16K): [in this window] [in a new window]   Figure 1. Characterization of a Mouse mAb Probe (cl 56) That Only Recognizes Tyrosine 14-Phosphorylated Caveolin-1 A tyrosine-phosphorylated caveolin-1 peptide (SEGHL(pY)TVPI, residues 9–18) was used to immunize mice and generate a phosphocaveolin-1-specific mAb probe. A, GST-caveolin-1 fusion proteins. A GST-fusion protein carrying the N-terminal domain of caveolin-1 (residues 1–101) was purified from normal bacteria (BL21) or from a bacterial strain harboring a tyrosine kinase (TKB1). Note that clone 56 recognized only tyrosine- phosphorylated caveolin-1 produced in the TKB1 strain, despite equal protein loading. B, Caveolin-1 Y14A mutant. Cos-7 cells were transiently transfected with caveolin-1 and c-Src, alone or in combination. Note that antiphosphocaveolin-1 IgG only recognizes caveolin-1 when it is coexpressed with c-Src. Tyrosine-phosphorylated caveolin-1 migrated as multiple bands, with a major band at approximately 24–25 kDa. Importantly, when tyrosine 14 is mutated to alanine (Y14A), this immunoreactivity is completely abolished. Immunoblotting with mAb 2297 that recognizes total caveolin-1 is shown as a control for equal loading. The mobility of caveolin-1 (Y14A) may be shifted due to a conformational change. C, Peptide competition.Cos-7 cells were transiently transfected with caveolin-1 and c-Src in combination and subjected to preparative SDS-PAGE. After transfer, the nitrocellulose sheet was cut into strips and incubated with antiphosphocaveolin-1 IgG alone or in combination with peptides. The sequences of these four caveolin-1-based peptides (PY14, Y14, PY100, and PY148) are detailed in Table 1.

View larger version (24K): [in this window] [in a new window]   Figure 2. Wild-Type and Mutationally Activated Src Phosphorylate Caveolin-1 on Tyrosine 14 A, IP/Western of caveolin-1 (WT and Y14A). Cos-7 cells were transiently cotransfected with caveolin-1 (WT or Y14A), alone or in combination with c-Src. Thirty-six hours post transfection, the cells were processed for immunoprecipitation. Total caveolin-1 was retrieved using IgG directed against the caveolin-1 C terminus (pAb H-97). Immunoprecipitates were then probed with a mAb that recognizes phosphotyrosine (mAb PY20). Note that when tyrosine 14 is mutated to alanine (Y14A), reactivity of caveolin-1 with PY20 is completely abolished. LC denotes the IgG light chain. B, Activated and kinase-dead c-Src mutants. Cos-7 cells were transiently transfected with caveolin-1 and c-Src [WT, activated (Y529F), and kinase-dead (K297R)]. Tyrosine 14- phosphorylated caveolin-1 was detected by Western blot analysis with antiphosphocaveolin-1 IgG. Activated c-Src phosphorylated caveolin-1 to a much greater extent than wild-type, and kinase-dead c-Src showed no activity. Immunoblotting with mAb 2297 that recognizes total caveolin-1 is shown as a control for equal loading. C, v-Src cells/WB. Lysates were prepared from normal and v-Src-transformed NIH 3T3 cells and subjected to immunoblot analysis. Note that antiphosphocaveolin-1 IgG recognizes tyrosine 14-phosphorylated caveolin-1 in NIH 3T3 cells that stably overexpress the v-Src. In contrast, little or no immunoreactivity was observed in normal NIH 3T3 cells. A comparison between normal and v-Abl-transformed NIH 3T3 cells is also shown. Each lane contains equal amounts of total protein. Immunoblotting with mAb 2297 that recognizes total caveolin-1 is also shown. D, v-Src cells/IP. v-Src-transformed NIH 3T3 cells were lysed and immunoprecipitated with an excess of mAb 2234 that recognizes total caveolin-1 (residues 1–21) or an excess of mAb 56 that recognizes phosphocaveolin-1 (residues 9–18). After SDS-PAGE and transfer to nitrocellulose, the amount of caveolin-1 precipitated was estimated by Western blotting with rabbit anticaveolin-1 IgG (N-20). Note that that only a small fraction ( 5%) of caveolin-1 undergoes phosphorylation on tyrosine 14.

To roughly estimate the percentage of total cellular caveolin-1 that undergoes phosphorylation on tyrosine 14, we employed an immunoprecipitation approach. v-Src- transformed NIH 3T3 cells were lysed and subjected to immunoprecipitation with mAb 2234, which recognizes total caveolin-1 (residues 1–21), or mAb 56, which recognizes phosphocaveolin-1 (residues 9–18). The amount of caveolin-1 precipitated was estimated by Western blotting with rabbit anticaveolin-1 IgG. Using this approach, Fig. 2D shows that only a fraction ( 5%) of total caveolin-1 undergoes phosphorylation on tyrosine 14.

Membrane attachment of c-Src is mediated in part by N-terminal myristoylation of the protein product (44). Interestingly, this modification is required for the targeting of c-Src to caveolae membrane domains (45). Caveolin-1 also undergoes lipid modification (22). However, the functional role of caveolin-1 palmitoylation remains largely unknown. Thus, we next examined the possible requirement of these lipid modifications for the tyrosine phosphorylation of caveolin-1.

To evaluate the role of c-Src myristoylation, Cos-7 cells were cotransfected with caveolin-1 and c-Src [wild-type (WT) or myristoylation-deficient (Myr-minus)]. Figure 3A shows that myristoylation of c-Src is required for phosphorylation of caveolin-1 on tyrosine 14. These results are consistent with the observation that myristoylation of c-Src is required for its targeting to caveolae (45).

View larger version (9K): [in this window] [in a new window]   Figure 3. Lipid Modification Is Required for Src-Induced Phosphorylation of Caveolin-1 on Tyrosine 14 A, Myristoylation minus c-Src. Cos-7 cells were cotransfected with caveolin-1 and c-Src (WT or Myr-minus). Note that myristoylation of c-Src is required for phosphorylation of caveolin-1 on tyrosine 14. B, Palmitoylation-deficient caveolin-1. To evaluate the role of caveolin-1 palmitoylation, we cotransfected Cos-7 cells with wild-type c-Src and caveolin-1 (WT or Pal-minus). Note that palmitoylation of caveolin-1 is required for or greatly facilitates phosphorylation of caveolin-1 on tyrosine 14. In panels A and B, immunoblotting with mAb 2297 that recognizes total caveolin-1 is shown as a control for equal loading.

Colocalization of Tyrosine 14-Phosphorylated Caveolin-1 with Markers of Focal Adhesions in Cells Expressing Activated Src
To examine the localization of tyrosine-phosphorylated caveolin-1, we cotransfected Cos-7 cells with caveolin-1 alone and in combination with either wild-type c-Src or activated c-Src (Y529F). These cells were then doubly immunostained with antiphosphocaveolin-1 mouse mAb (cl 56) and an anticaveolin-1 rabbit polyclonal antibody (pAb) (N-20). These bound primary antibodies were visualized by using distinctly tagged fluorescent secondary antibodies (see Materials And Methods).

Figure 4 shows the distribution of caveolin-1 phosphorylated on tyrosine 14. In cells transfected with caveolin-1 alone, little or no immunostaining with antiphosphocaveolin-1 was observed (Fig. 4A). In cells cotransfected with caveolin-1 plus wild-type c-Src, immunostaining with antiphosphocaveolin-1 appeared as large fluorescent dots in the center and along the cell periphery (Fig. 4B). Immunostaining with anticaveolin-1 also appeared punctate, but the dots were of a much smaller size. These two labeling patterns appeared distinct, suggesting that tyrosine-phosphorylated caveolin-1 is present in a different region of the cell or that only a subpopulation of caveolae are tyrosine phosphorylated. In cells cotransfected with caveolin-1 plus activated c-Src (Y529F), immunostaining with antiphosphocaveolin-1 also appeared as large dots, but these dots were confined to the cell periphery and appeared to coincide with focal contacts or adhesions (Fig. 4C). A very similar staining pattern was observed in v-Src-transformed NIH 3T3 cells (Fig. 5A).

View larger version (69K): [in this window] [in a new window]   Figure 4. Localization of Tyrosine 14-Phosphorylated Caveolin-1 in Cos-7 Cells Transiently Transfected with c-Src To examine the localization of tyrosine-phosphorylated caveolin-1, we cotransfected Cos-7 cells with caveolin-1 and either WT or activated c-Src. Cells were doubly immunostained with antiphosphocaveolin-1 mouse mAb (cl 56; at left) and an anticaveolin-1 rabbit pAb (N-20; at right). Bound primary antibodies were visualized by using distinctly tagged fluorescent secondary antibodies. A, Caveolin-1 alone.In Cos-7 cells transfected with caveolin-1 alone, little or no immunostaining with antiphosphocaveolin-1 was observed. B, Caveolin-1 plus wild-type c-Src. In cells cotransfected with caveolin-1 plus WT c-Src, immunostaining with antiphosphocaveolin-1 appeared as large fluorescent dots in the central region of the cell and along the cell periphery. C, Caveolin-1 plus activated c-Src. In cells cotransfected with caveolin-1 plus activated c-Src (Y529F), immunostaining with antiphosphocaveolin-1 appeared as large dots confined to the cell periphery that were reminiscent of focal contacts or adhesions. In panels A-C, immunostaining with anticaveolin-1 IgG appeared punctate (right) and did not coincide with the labeling observed with antiphosphocaveolin-1.

View larger version (29K): [in this window] [in a new window]   Figure 5. Tyrosine 14-Phosphorylated Caveolin-1 Is Localized in Close Proximity to Focal Adhesions in NIH 3T3 Cells Stably Expressing v-Src Cells were doubly immunostained with two distinct primary antibodies as detailed below. Bound primary antibodies were visualized by using distinctly tagged fluorescent secondary antibodies. In panels A, B, and D, images were acquired with a MR600 confocal fluorescence microscope (Bio-Rad Laboratories, Inc.). In panels C and E, images were acquired with a IX80 microscope (Olympus Corp.) with a 60x Plan Neofluar objective and a Photometrics cooled CCD camera with a 35-mm shutter. A, Antiphosphocaveolin-1 (mAb) and anticaveolin-1 (pAb). Note that staining with antiphosphocaveolin-1 appeared as large dots, but these dots were confined to the cell periphery and appeared to coincide with focal contacts or adhesions. B, Antiphosphocaveolin-1 (mAb) and antiphospho tyrosine (pAb). Note that tyrosine-phosphorylated caveolin-1 is colocalized with the major sites of tyrosine phosphorylation. These sites of tyrosine phosphorylation activity are known to correspond to focal adhesions (48 ). C, Anti-phospho-caveolin-1 (mAb) and antipaxillin. Note that caveolin-1 and paxillin colocalize to a significant extent. These results suggest that caveolae in close proximity to focal adhesions are preferentially phosphorylated in v-Src-transformed cells. D, Anticaveolin-1 (pAb) and antiphosphotyrosine (PY20 mAb). Note that little or no colocalization is observed. These results indicate that the bulk of total caveolin-1 is not localized in proximity to the major sites of tyrosine phosphorylation in vivo. E, Color overlay. A color version of panel C is shown along with the merged image to better demonstrate the colocalization of phosphocaveolin-1 and paxillin.

In contrast, when v-Src-transformed NIH 3T3 cells were double-labeled with anticaveolin-1 (N-20 pAb) and antiphosphotyrosine (PY20 mAb), little or no colocalization was observed (Fig. 5D). These results indicate that in v-Src-transformed cells the bulk of total caveolin-1 is not localized in proximity to the major sites of tyrosine phosphorylation in vivo. This is consistent with the observation that only a fraction of total cellular caveolin-1 ( 5%) is tyrosine phosphorylated in v-Src-transformed cells (Fig. 2D).

Tyrosine-Phosphorylated Caveolin-1 Correctly Forms High Molecular Mass Oligomers and Is Targeted to Caveolae-Enriched Membrane Domains
As tyrosine-phosphorylated caveolin-1 was localized in close proximity to focal adhesions, we next assessed the biochemical properties of tyrosine-phosphorylated caveolin-1 using established assay systems. These approaches have been used previously to characterize the properties of total caveolin-1. Caveolin-1 normally forms stable high molecular mass homooligomeric complexes (7, 18). Once these homooligomers reach the plasma membrane, they become Triton insoluble due to their incorporation into caveolae membranes (49). This resistance to detergent solubilization is thought to reflect the local lipid microenvironment in which these caveolin homooligomers are embedded. In contrast, caveolin-1 associated with the Golgi complex remains Triton soluble (50). Thus, we next examined the effects of the tyrosine phosphorylation of caveolin-1 on its 1) Triton insolubility; 2) oligomeric state; and 3) caveolar targeting, using normal and v-Src- transformed NIH 3T3 cells.

In both normal and v-Src-transformed NIH 3T3 cells, total caveolin-1 was >95% Triton insoluble (not shown), formed high molecular mass oligomers of the correct size, and was targeted to caveolae-enriched membrane fractions (Fig. 6, A and B). Interestingly, virtually identical results were obtained with caveolin-1 phosphorylated on tyrosine 14, indicating that caveolin-1 remains caveolae associated even after tyrosine phosphorylation in v-Src-transformed cells. As the subcellular distribution of tyrosine- phosphorylated caveolin-1 coincides with focal adhesions by fluorescence microscopy (Fig. 6), these biochemical results are consistent with the idea that caveolae in close proximity to focal adhesions are preferentially targeted for phosphorylation in v-Src- transformed cells.

View larger version (15K): [in this window] [in a new window]   Figure 6. Tyrosine 14-Phosphorylated Caveolin-1 Correctly Forms High Molecular Mass Oligomers and Is Targeted to Caveolae-Enriched Membrane Domains In both panels, tyrosine phosphorylated caveolin-1 was detected with mAb 56 and total caveolin-1 was visualized using mAb 2297. A, Velocity gradient analysis. Normal and v-Src-transformed NIH 3T3 cells were solubilized, loaded atop a 5–40% sucrose density gradient, and subjected to centrifugation for 10 h. Twelve fractions were recovered and a 20 µl aliquot from each fraction was analyzed by SDS-PAGE/Western blotting. Arrows mark the positions of molecular mass standards. Note that caveolin-1 behaves as a high molecular mass oligomer of approximately 150–300 kDa. B, Caveolar targeting. Normal and v-Src-transformed NIH 3T3 cells were homogenized in a buffer containing 1% Triton X-100 and subjected to sucrose density gradient centrifugation. Twelve 1-ml fractions were collected, and an aliquot of each fraction ( 20 µl) was analyzed by SDS-PAGE/Western blotting. As expected, caveolin-1 is highly enriched in fractions 4–5, which represent the caveolae-enriched membrane fractions. Note that both total caveolin-1 and tyrosine 14- phosphorylated caveolin-1 form high molecular mass oligomers of the correct size and are targeted to caveolae-enriched membrane fractions in normal and v-Src-transformed cells.

This insulin-stimulated tyrosine phosphorylation of caveolin is thought to occur via an endogenous member of the Src family of tyrosine kinases (41). However, it remains unknown which tyrosine residue is phosphorylated in response to insulin stimulation. Also, as caveolins-1 and -2 form a tight heterooligomeric complex and stably coimmunoprecipitate (18, 26, 56, 57), it remains unclear whether caveolin-1 or caveolin-2 is actually the target of tyrosine phosphorylation in adipocytes.

To address these issues, we treated 3T3-L1 adipocytes with insulin or a variety of other growth factors. Figure 7 shows that a brief treatment (10 min) of 3T3-L1 adipocytes with insulin dramatically stimulated phosphorylation of caveolin-1 on tyrosine 14, while the other factors had little or no effect (see panels A and C). Interestingly, 3T3-L1 adipocytes showed an increased basal level of tyrosine phosphorylation of caveolin-1, as compared with Cos-7 cells or NIH 3T3 cells. Virtually identical results were obtained with 3T3-L1 fibroblasts, indicating that this event also occurs in the preadipocyte (Fig. 7A). Tyrosine phosphorylation of caveolin-1 was concentration dependent and increased from 5–150 nM insulin (not shown). Insulin-stimulated phosphorylation of caveolin-1 on tyrosine 14 was apparent after only 1 min of treatment, reached maximal levels at 5 min, and declined to basal levels by 120 min (Fig. 7B). These results are consistent with the idea that tyrosine phosphorylation of caveolin-1 is an early event in insulin receptor signal transduction.

View larger version (24K): [in this window] [in a new window]   Figure 7. Insulin-Stimulated Phosphorylation of Caveolin-1 on Tyrosine 14 in 3T3-L1 Adipocytes In panels A–C, cells were lysed and subjected to immunoblot analysis with antiphosphocaveolin-1 IgG. Immunoblotting with mAb 2297 that recognizes total caveolin-1 is shown as a control for equal loading. A, Growth-factor stimulation. 3T3-L1 adipocytes (left) or fibroblasts (right) were serum starved for 3 h and subsequently treated with insulin (150 nM) or a variety of other growth factors (PDGF (10 ng/ml), bFGF (5 ng/ml), TNF (10 nM), and IL6 (5 nM)). Note that a brief treatment (10 min) of 3T3-L1 adipocytes with insulin dramatically stimulated phosphorylation of caveolin-1 on tyrosine 14, while the other factors had little or no effect (left). Virtually identical results were obtained with 3T3-L1 preadipocytes (right). B, Insulin time course. 3T3-L1 adipocytes were serum starved for 3 h and subsequently treated with insulin. Note that insulin (150 nM)-stimulated tyrosine phosphorylation of caveolin-1 was apparent after only 1 min of treatment, reached maximal levels at 5 min, and declined to basal levels by 120 min. C, Insulin vs. EGF stimulation. 3T3-L1 adipocytes were serum starved for 3 h and subsequently treated with EGF (100 ng/ml) or insulin (150 nM). Note that a brief treatment (5 min) of 3T3-L1 adipocytes with insulin stimulated phosphorylation of caveolin-1 on tyrosine 14, while treatment with EGF had no effect.

EGF-Stimulated Phosphorylation of Caveolin-1 on Tyrosine 14 in A431 Cells
A431 cells are a human epidermoid carcinoma-derived cell line that has been used extensively to study EGF-mediated signal transduction. As A431 cells are known to express both EGF receptor (EGF-R) (58) and caveolin-1 (21, 59), we used these cells to evaluate the effects of EGF stimulation on the tyrosine phosphorylation of caveolin-1. For this purpose, A431 cells were serum starved and treated with EGF (100 ng/ml) for various times. As a positive control for these studies, we used an antibody that only detects the activated phosphorylated form of the EGF-R (58). Figure 8A shows that a brief treatment (5 min) of A431 cells with EGF activated the EGF-R and dramatically stimulated phosphorylation of caveolin-1 on tyrosine 14. This is in contrast to our results with 3T3-L1 adipocytes (Fig. 7C), which do not show tyrosine phosphorylation of caveolin-1 in response to EGF, despite the fact that these cells are known to express the EGF-R and are EGF responsive. Thus, growth factor-stimulated tyrosine phosphorylation of caveolin-1 may be dependent on cell-type specific coupling factors or simply dependent on the relative expression levels of different growth factor receptors in a given cell type.

View larger version (94K): [in this window] [in a new window]   Figure 8. EGF-Stimulated Phosphorylation of Caveolin-1 on Tyrosine 14 in A431 Cells A, EGF time course. A431 cells were serum-starved for 12–16 h and subsequently treated with EGF (100 ng/ml) for 0–4 h. Cells were lysed and subjected to immunoblot analysis with antiphosphocaveolin-1 IgG. As a positive control, we used an antibody that only detects the activated phosphorylated form of the EGF-R (mAb 74). Note that a brief treatment (5 min) of A431 cells with EGF dramatically activated EGF-R and stimulated the phosphorylation of caveolin-1 on tyrosine 14. Immunoblotting with phospho-independent antibodies to EGF-R (mAb 13) and caveolin-1 (mAb 2297) was used to visualize total EGF-R and total caveolin-1 levels. Each lane contains an equal amount of total protein. B, Immunolocalization. Confluent A431 cells were serum starved and incubated in the presence or absence of EGF (100 ng/ml) for 5 min. Note that tyrosine-phosphorylated caveolin-1 accumulated at the plasma membrane and was highest in areas of cell-cell contact. Importantly, little or no immunostaining was observed using antiphosphocaveolin-1 IgG if A431 cells were not treated with EGF. C, Colocalization with paxillin. Subconfluent A431 cells were serum-starved and subsequently incubated in the presence of EGF (100 ng/ml) for 5 min. Double labeling with antiphosphocaveolin-1 and antipaxillin revealed that caveolin-1 and paxillin colocalize to a significant extent. Arrowheads point to areas of colocalization. These results suggest that caveolae in close proximity to focal adhesions are preferentially phosphorylated in EGF-stimulated A431 cells.

Vanadate Treatment Induces the Accumulation of Tyrosine 14-Phosphoryated Caveolin-1 in Normal NIH 3T3 Cells
It remains unknown what ligands might stimulate tyrosine phosphorylation of caveolin-1 in other cell types, such as NIH 3T3 cells. For example, treatment of NIH 3T3 cells with various growth factors [EGF, PDGF, and basic fibroblast growth factor (bFGF)] had no apparent effect on the tyrosine phosphorylation of caveolin-1 (not shown). To investigate whether caveolin-1 undergoes tyrosine phosphorylation in normal NIH 3T3 cells, these cells were treated with vanadate (100 µM), a tyrosine phosphatase inhibitor. If caveolin-1 undergoes tyrosine phosphorylation, vanadate should prevent dephosphorylation and allow detection of accumulated tyrosine-phosphorylated intermediates. Figure 9A shows that treatment with vanadate for increasing times leads to the accumulation of tyrosine 14- phosphorylated caveolin-1. As in v-Src-transformed cells, several caveolin-1 bands of approximately 22–28 kDa were apparent (compare Figs. 2C and 9A).

View larger version (14K): [in this window] [in a new window]   Figure 9. Vanadate-Treatment Induces the Accumulation of Tyrosine 14-Phosphoryated Caveolin-1 in Normal NIH 3T3 Cells To investigate whether caveolin-1 undergoes tyrosine phosphorylation in normal NIH 3T3 cells, these cells were treated with vanadate (100 µM), a tyrosine phosphatase inhibitor. A, Western blot analysis. Note that treatment with vanadate for increasing times leads to the accumulation of tyrosine 14-phosphorylated caveolin-1. As in v-Src- transformed cells, several caveolin-1 bands of approximately 22–28 kDa are apparent. B, Immunofluorescence. NIH 3T3 cells were treated with vanadate for 2 h or left untreated. Note that tyrosine-phosphorylated caveolin-1 accumulated at the plasma membrane and was highest in areas of cell-cell contact (upper panels). Importantly, little or no immunostaining was observed using antiphosphocaveolin-1 IgG if normal NIH 3T3 cells were not treated with vanadate. In contrast, immunostaining of vanadate-treated NIH 3T3 cells with anti-caveolin-1 IgG demonstrated that caveolin-1 was predominantly localized to a perinuclear compartment and at areas of cell-cell contact (lower panels). Thus, tyrosine 14-phosphorylated caveolin-1 accumulated predominantly at the plasma membrane and did not strictly follow the distribution of total caveolin-1 in vanadate-treated NIH 3T3 cells. Images were acquired with an MR600 confocal fluorescence microscope(Bio-Rad Laboratories, Inc.).

Purified Caveolae-Enriched Membranes Contain a Kinase That Phosphorylates Caveolin-1 on Tyrosine 14 in Vitro
Purified caveolae membranes are dramatically enriched in caveolin-1 and have been shown to contain tyrosine kinase activity (6, 27, 41, 60, 61). Also, a number of Src-family tyrosine kinases (c-Src, c-Yes, Lyn, Fyn, Lck, and c-Fgr) and receptor tyrosine kinases (Ins-R, EGF-R, and PDGF-R) copurify with caveolae and caveolin-1 under these conditions (1, 27, 28, 30, 31, 61, 62, 63, 64, 65, 66, 67, 68). Thus, we examined the ability of caveolin-1 to be phosphorylated on tyrosine 14 using purified caveolae-enriched membrane domains.

Caveolae-enriched domains were purified from murine lung tissue using an established protocol (28) and incubated in kinase reaction buffer in the presence or absence of exogenous ATP (1 mM). Figure 10A (left panel) shows that addition of ATP dramatically induced phosphorylation of caveolin-1 on tyrosine 14. Thus, a kinase activity with the same specificity as is observed in vivo is present within purified caveolae membranes and copurifies with caveolin-1. Other tyrosine-phosphorylated proteins were visualized by immunoblotting with an antiphosphotyrosine mAb (PY20); the major tyrosine-phosphorylated proteins appeared in the 50- to 60-kDa and the 200-kDa range, consistent with the autophosphorylation of Src-family tyrosine kinases and receptor tyrosine kinases, respectively (Fig. 10A, right panel). [It should be noted that a 22- to 25-kDa band corresponding to caveolin-1 was also detectable with PY20 on longer exposures (not shown)].

View larger version (14K): [in this window] [in a new window]   Figure 10. Purified Caveolae-Enriched Membranes Contain a Kinase That Phosphorylates Caveolin-1 on Tyrosine 14 in Vitro A, In vitro kinase assay. Caveolae-enriched domains were purified from murine lung tissue using an established protocol (28 ) and incubated in kinase reaction buffer in the presence or absence of exogenous ATP (1 mM). After 10 min at 25 C, the reaction was halted by the addition of SDS sample buffer/boiling and samples were subjected to Western blotting. Note that addition of ATP dramatically induced phosphorylation of caveolin-1 on tyrosine 14 (left panel). The total pattern of tyrosine-phosphorylated proteins was visualized by immunoblotting with an antiphosphotyrosine mAb (PY20) (right panel). Immunoblotting with mAb 2297 that recognizes total caveolin-1 is shown as a control for equal loading (middle panel). B, Inhibitor profile. Note that pretreatment of caveolae-enriched membranes with either AG1478 or A9 (5 µM) had little or no effect (upper panel). In contrast, pretreatment with PP2 (5 µM) dramatically inhibited the phosphorylation of caveolin-1 on tyrosine 14. Immunoblotting with mAb 2297 that recognizes total caveolin-1 is shown as a control for equal loading (lower panel).

Identification of Grb7 as a pY14-Caveolin-1 Binding Partner in Vitro and in Vivo
One known function of tyrosine phosphorylation is to confer binding to SH2 domain- containing proteins. To identify SH2 domain proteins that bind to tyrosine-phosphorylated caveolin-1, we used an established in vitro binding approach. Briefly, we prepared nonphosphorylated and tyrosine-phosphorylated caveolin-1 from bacteria (Fig. 1A; see Materials and Methods). These purified GST-Cav-1 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101) fusion proteins were then incubated with lysates prepared from normal NIH 3T3 cells. After extensive washing, the bound material was subjected to Western blot analysis with a panel of antibodies directed against SH2 domain-containing proteins (listed in Table 2).

View larger version (24K): [in this window] [in a new window]   Figure 11. Tyrosine 14-Phosphorylated Caveolin-1 Binds Grb7 in a Phosphorylation-Dependent Manner A, Grb7 binding in vitro. Nonphosphorylated and tyrosine-phosphorylated GST-Cav-1 (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 ) fusion proteins were prepared and incubated with cell lysates derived from normal NIH 3T3 cells. After binding, washing, and elution, the eluates were subjected to immunoblot analysis with antibodies directed against 20 different SH2 domain-containing proteins (listed in Table 2). Left panel, Note that only the binding of Grb7 was observed. Importantly, Grb7 bound to tyrosine-phosphorylated GST-Cav-1 (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 ), but not to GST alone, or nonphosphorylated GST-Cav-1 (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 ). Right panel, Other closely related proteins, such as Grb2 and Grb14, did not show any binding activity. B, Grb7 binding in vivo. Left panel, Cos-7 cells were co-transfected with cDNAs encoding caveolin-1 and Grb7, in the presence of c-Src WT or its corresponding empty vector (pUSE). Forty-eight hours post transfection, lysates were prepared and subjected to immunoprecipitation with antibodies directed against Grb7. These immunoprecipitates were then subjected to Western blot analysis with anticaveolin-1 IgG or antiphosphocaveolin-1 IgG. Note that both antibodies detect caveolin-1 coimmunoprecipitating with Grb7, but not with beads alone or an irrelevant IgG; and this binding occurs only in cells cotransfected with c-Src WT. Right panel, Note that this binding is strictly dependent on tyrosine 14, as caveolin-1 (Y14A) fails to coimmunoprecipitate with Grb7. C, Importance of tyrosine 14. Left panel, Cos-7 cells were cotransfected with cDNAs encoding caveolin-1 and Grb-7, in the presence of c-Src WT. Lysates were prepared and subjected to immunoprecipitation with antibodies directed against Grb7. Immunoprecipitates were then subjected to Western blotting with antiphosphocaveolin-1 IgG. Before immunoprecipitation, competing peptides were added to these lysates (detailed in Table 1). Note that the PY14 peptide completely blocks this interaction, while the corresponding nonphosphorylated peptide (Y14) or an irrelevant phosphopeptide (PY100) has no effect. Right panel, Cos-7 cells were transiently transfected with the cDNA encoding Grb-7. Lysates were prepared and incubated with biotinylated phosphopeptides prebound to streptavidin beads. After washing, these precipitates were subjected to Western blotting with anti-Grb7 IgG. Note that the caveolin-1-derived phosphopeptide containing tyrosine 14 (PY14) effectively pulls down Grb7, while another irrelevant caveolin-1-derived phosphopeptide containing tyrosine 100 (PY100) does not.

To further investigate the specific role of caveolin-1 tyrosine 14, we next used a peptide-based approach. Cos-7 cells were cotransfected with the cDNAs encoding caveolin-1 and Grb-7, in the presence of c-Src. Lysates were prepared and competing peptides were added (detailed in Table 1), and they were immunoprecipitated with antibodies directed against Grb7. These immunoprecipitates were subjected to Western blotting with antiphosphocaveolin-1 IgG. Figure 11C (left panel) shows that addition of the PY14 peptide completely blocks this interaction, while the addition of the corresponding nonphosphorylated peptide (Y14) or an irrelevant phosphopeptide (PY100) has no effect. These results again implicate phosphorylated tyrosine 14 in this binding event.

To assess whether phosphotyrosine 14 and its surrounding sequence is sufficient for Grb7 binding, Cos-7 cells were transiently transfected with the cDNA encoding Grb-7. Lysates were then incubated with biotinylated phosphopeptides bound to streptavidin beads. After washing, these precipitates were subjected to Western blot analysis with anti- Grb7 IgG. Figure 11C (right panel) shows that a caveolin-1-derived phosphopeptide containing tyrosine 14 (PY14) effectively pulls down Grb7, while another irrelevant caveolin-1-derived phosphopeptide (PY100) does not. Thus, caveolin-1 phosphotyrosine 14 and its surrounding sequence are sufficient for Grb7 binding.

Binding of Grb7 to Tyrosine 14-Phosphorylated Caveolin-1 Functionally Stimulates Anchorage-Independent Growth and Cell Migration
What is the function of tyrosine-phosphorylated caveolin-1 (Y14) ? As tyrosine-phosphorylated caveolin-1 is localized at focal adhesions, we speculated that it might play a role in regulating anchorage-dependent growth and/or cell migration. To test this hypothesis, we used 293T cells because they lack endogenous caveolin-1 expression and they undergo transfection with high efficiency, a prerequisite for these cotransfection studies. In this cellular context, we compared the activity of wild-type caveolin-1 with a mutant form of caveolin-1 (Y14A) that is unable to undergo tyrosine phosphorylation at residue 14.

Anchorage-Independent Growth Studies
Figure 12A shows that expression of caveolin-1 alone inhibited foci formation in 293T cells. This is consistent with previous reports showing that recombinant expression of caveolin-1 in other cell types inhibits anchorage-independent growth (72, 73, 74). Expression of c-Src alone had no effect on foci formation; it has been previously shown that overexpression of c-Src is not sufficient to mediate cell transformation (75). In contrast, coexpression of c-Src and caveolin-1 stimulated foci formation by approximately 2-fold, as compared with mock-transfected or cells transfected with Src alone. These results suggest that tyrosine phosphorylation of caveolin-1 can stimulate anchorage-independent growth.

View larger version (11K): [in this window] [in a new window]   Figure 12. Binding of Grb7 to Tyrosine 14-Phosphorylated Caveolin-1 Stimulates Both Anchorage-Independent Growth and Cell Migration 293T cells were cotransfected with the cDNAs encoding c-Src, Grb7, caveolin-1 (WT), and caveolin-1 (Y14A), alone or in combination. Cells were then subjected to focus formation or cell migration assays. A, Anchorage-independent growth. Note that coexpression of c-Src and caveolin-1 stimulated foci formation by approximately 2-fold, as compared with mock-transfected cells or cells transfected with Src alone. In addition, coexpression of c-Src, caveolin-1 (WT), and Grb7 dramatically stimulated foci formation by approximately 3.5-fold, as compared with mock-transfected cells, and by approximately 7-fold as compared with cells cotransfected with c-Src and Grb7. In striking contrast, coexpression of c-Src, caveolin-1 (Y14A), and Grb7 had no effect on foci formation. B, Cell migration. Note that coexpression of c-Src, caveolin-1 (WT), and Grb7 dramatically stimulated cell migration by approximately 2- to 3-fold, as compared with mock-transfected cells, cells transfected with caveolin-1 alone, or cells cotransfected with c-Src and Grb7. In contrast, coexpression of c-Src, caveolin-1 (Y14A), and Grb7 had little or no effect on cell migration.

Cell Migration Studies
293T cells are known to express endogenous EGF-R and to undergo EGF-stimulated cell migration. Thus, we next assessed the effect of the Src/Cav-1/Grb7 signaling cassette on this process (Fig. 12B). Interestingly, coexpression of c-Src, caveolin-1 (WT), and Grb7 dramatically stimulated cell migration by approximately 2- to 3-fold, as compared with mock-transfected cells, cells transfected with caveolin-1 alone, or cells transfected with c-Src and Grb7 alone. In contrast, coexpression of c-Src, caveolin-1 (Y14A), and Grb7 had little or no effect on cell migration, as compared with mock-transfected cells, and behaved the same as cells transfected with c-Src and Grb7 alone. Thus, binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 augments both anchorage-independent growth and EGF-stimulated cell migration in 293T cells.

It should also be noted that expression of caveolin-1 alone (either WT or Y14A) had little or no effect on cell migration in 293T cells. This is in contrast to our previous results employing MTLn3 cells, a metastatic rat mammary adenocarcinoma cell line (74). In MTLn3 cells, caveolin-1 expression blocked EGF-stimulated cell migration (74). Thus, the negative regulatory effect of caveolin-1 on cell migration may be cell type specific.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The oncogene v-Src arose by viral transduction of the normal cellular gene c-Src (76, 77). Thus, it is thought that viral tyrosine kinases largely induce transformation by intercepting cell-regulatory mechanisms that are normally under the control of tyrosine phosphorylation. In support of this notion, v-Src and c-Src appear to differ primarily in enzymatic activity, but not in their substrate specificity (78, 79). This difference in enzymatic activity can be attributed to the loss of tyrosine residue 527 within v-Src (residue 529 in human c-Src). Phosphorylation at this C-terminal site within c-Src by CSK normally inactivates c-Src (80, 81).

Few physiologically relevant v-Src substrates are known. Caveolin-1 is one of these substrates (11, 12, 13, 40). For example, caveolin-1 copurifies as a heterooligomeric complex with c-Src and other Src-family tyrosine kinases (27, 28, 30, 35). This is consistent with the general idea that v-Src phosphorylates the normal targets of c-Src or related Src-family tyrosine kinases, but in an unregulated fashion.

Here, we studied the phosphorylation of caveolin on tyrosine in vivo by employing a novel phosphospecific mAb probe that selectively recognizes tyrosine 14-phosphorylated caveolin-1. We reconstituted this event by cotransfecting caveolin-1 with c-Src. We observed that myristoylation of c-Src and palmitoylation of caveolin-1 are both required for Src-induced phosphorylation of caveolin-1 on tyrosine 14. In cells transfected with activated forms of Src [either c-Src (Y529F) or v-Src], tyrosine-phosphorylated caveolin-1 was localized mainly in close proximity to focal adhesions, the major cellular sites of tyrosine kinase-mediated signal transduction.

We also evaluated the tyrosine phosphorylation of caveolin-1 in other cell types. In 3T3-L1 adipocytes, insulin dramatically increased the phosphorylation of caveolin-1 on tyrosine 14, with maximal stimulation at 5 min of treatment. In A431 cells, EGF stimulation greatly increased the tyrosine phosphorylation of caveolin-1, which was localized near focal adhesions. Incubation of NIH 3T3 cells with vanadate (a tyrosine phosphatase inhibitor) drove the accumulation of tyrosine-phosphorylated caveolin-1. Also, purified caveolae membranes contain a kinase activity that phosphorylates caveolin-1 on tyrosine 14 in vitro. This phosphorylation event was inhibited by a selective inhibitor of Src-family tyrosine kinases (PP2). Importantly, these results demonstrate that constitutive (via activated Src) and regulated (via insulin, EGF, or vanadate treatment) phosphorylation of caveolin-1 occur at the same site, i.e. tyrosine 14, in vivo.

Caveolin-1 exists as a high molecular mass oligomer containing approximately 14–16 individual caveolin molecules (23, 25), and these caveolin oligomers have the capacity to self-associate into larger complexes (23). By analogy with other known tyrosine phosphorylation events, we speculated that tyrosine-phosphorylated caveolin-1 could potentially serve as an oligomeric docking-site for SH2-domain signaling molecules (23, 42)—much like activated growth factor receptors that oligomerize, undergo tyrosine phosphorylation, and recruit SH2 domain-containing proteins to the cytoplasmic surface of the plasma membrane (Fig. 13). In support of this hypothesis, we show here that phosphorylation of caveolin-1 on tyrosine 14 functionally confers binding to Grb7, an SH2 domain-containing adaptor protein. Of the 20 phosphotyrosine-binding proteins we evaluated, Grb7 was the only one that showed binding activity toward recombinant tyrosine-phosphorylated GST-caveolin-1.

View larger version (21K): [in this window] [in a new window]   Figure 13. Schematic Diagram Summarizing the Src-Induced Phosphorylation of Caveolin-1 on Tyrosine 14 In step 1, lipid modification of both c-Src and caveolin-1 is required for maintaining their close proximity. In step 2, c-Src phosphorylates caveolin-1 on tyrosine 14. See text for details. Note that caveolin-1 forms a high molecular mass homooligomer of approximately 350 kDa that contains about 14–16 caveolin-1 monomers (23 36 46 ). Thus, a single caveolin-1 homooligomer could contain up to 48 palmitoyl acyl chains per oligomer (3 x 16 = 48) (22 ). For simplicity, caveolin-1 is drawn as a dimeric molecule. As such, tyrosine 14-phosphorylated caveolin-1 may function as a large oligomeric docking site for SH2 or PTB domain containing adaptor molecules, such as Grb7. KD, Kinase domain; PTB (phosphotyrosine binding).

Recent evidence indicates that Grb7 contains an SH2 domain and a PTB domain (phosphotyrosine-binding domain) (87). As such, Grb7 could act as a divalent bridge to link caveolin-1 to other tyrosine-phosphorylated proteins. As other Grb7-binding proteins include growth factor receptor tyrosine kinases (83) and FAK (focal adhesion kinase) (89), this might explain the colocalization of tyrosine 14-phosphorylated caveolin-1 with focal adhesions and total phosphotyrosine immunoreactivity in intact cells. This proposal is consistent with previous reports demonstrating that caveolin-1 coimmunoprecipitates with EGF-R (62) and integrin subunits (90). Also, our findings may explain recent data showing that caveolin-1 is a positive coupling factor in integrin-mediated signaling (presumably near focal adhesions) involving Fyn, a Src family tyrosine kinase (91). However, these investigators examined neither the phosphorylation state of caveolin-1 nor the cellular localization of caveolin-1 under these conditions.

Using a cotransfection approach, we show here that caveolin-1 can cooperate with c-Src and Grb7 to augment 1) anchorage-independent growth; and 2) EGF-stimulated cell migration. This effect is strictly dependent on tyrosine phosphorylation of caveolin-1, as mutant caveolin-1 (Y14A) does not show such cooperativity. These current results may serve to reconcile the two conflicting views of caveolin-1 that have appeared in the literature. Evidence has been presented that caveolin-1 is down-regulated during cell transformation and that replacement of caveolin-1 expression can reverse the transformed phenotype, suggesting that caveolin-1 behaves as a tumor suppressor (67, 72, 73, 74, 92, 93, 94, 95). This is also consistent with biochemical evidence showing that the caveolin-scaffolding domain can function as a negative regulator of a variety of mitogenic signaling molecules (3). On the other hand, caveolin-1 levels have been shown to be elevated in certain human tumors and to positively correlate with metastasis (96); in addition, other evidence suggests that caveolin-1 may function as a positive regulator in integrin signaling (90, 91, 97). These two, seemingly mutually exclusive, effects of caveolin-1 may be simply mediated by different regions of the caveolin-1 molecule and may be dependent on the levels of other molecules that are coexpressed with caveolin-1, such as c-Src and Grb7. More specifically, the caveolin-1 scaffolding domain could confer the transformation suppressor activity, while tyrosine phosphorylation of caveolin-1 at residue 14 could confer binding to SH2 domain-containing proteins (such as Grb7) and subsequent growth- stimulatory or oncogenic activity. In this way, caveolin-1 would be able to function both as a negative and positive regulator of signaling and cell transformation.

Recently, another paper appeared describing the generation of a rabbit polyclonal antibody that recognizes tyrosine 14-phosphorylated caveolin-1 (98). These authors primarily studied the tyrosine phosphorylation of caveolin-1 in a fibroblastic cell line stably transfected with a temperature-sensitive form of v-Src. Moreover, they performed immunoelectron microscopy with their phosphospecific rabbit polyclonal antibody (98). Interestingly, they showed that the large dots observed by immunofluorescence ultrastructurally correspond to clusters of caveolae (98). These results are consistent with our biochemical fractionation data (Fig. 6) showing that caveolin-1 is still confined to caveolae-enriched membrane fractions after phosphorylation on tyrosine 14. However, they did not report an association of caveolin-1 with focal adhesions.

In summary, our current study differs significantly from the work of Nomura and colleagues (98) in that we 1) developed a mouse mAb that recognizes tyrosine 14- phosphorylated caveolin-1; 2) showed that antigenic peptide competition or mutation of tyrosine 14 blocks the binding of our antibody to caveolin-1; 3) evaluated the effect of wild-type, activated, and kinase-dead c-Src; 4) evaluated the role of caveolin-1 palmitoylation and c-Src myristoylation in this process; 5) showed localization of caveolin-1 to focal adhesions in cells transfected with activated forms of Src or after growth factor stimulation; 6) evaluated the phosphorylation of caveolin-1 on tyrosine 14 in insulin signaling in adipocytes and EGF signaling in epidermoid cells; 7) used purified caveolae membrane domains to reconstitute this phosphorylation event in vitro; 8) identified a protein (Grb7) that specifically binds to tyrosine-phosphorylated caveolin-1; and 9) showed that Grb7 binding to tyrosine-phosphorylated caveolin-1 functionally augments anchorage-independent growth and EGF-stimulated cell migration.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Materials
The cDNAs for caveolin-1, -2, and -3 subcloned into the cytomegalovirus (CMV)-based vector, pCB7, were as we described previously (7, 17, 18, 19, 20, 27). The cDNA encoding nonpalmitoylated caveolin-1 was as described (22), except that it was subcloned into pCB7 (47). The cDNAs encoding human c-Src [WT, activated (Y529F), and kinase-dead (K297R)] in the pUSEamp CMV-based vector were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). cDNAs encoding chicken c-Src (WT and Myr-minus) and NIH 3T3 cells expressing v-Src were provided by Drs. Shalloway and Dehn (Cornell University, Ithaca, NY) (75). Antibodies and their sources were as follows: anticaveolin-1 IgG (mAb 2297); anti-EGF-R IgG (mAb 13); anti-activated EGF-R IgG (mAb 74) (all BD Transduction Laboratories, Inc., Lexington, KY); anti-Myc epitope IgG (mAb 9E10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anticaveolin-1 IgG (N-20 pAb; rabbit antipeptide antibody directed against caveolin-1 residues 2–21; Santa Cruz Biotechnology, Inc.); antiphosphotyrosine rabbit pAb (BD Transduction Laboratories, Inc.); antiphosphotyrosine mouse mAb (PY20; BD Transduction Laboratories, Inc.); antipaxillin mAb preconjugated to LRSC (BD Transduction Laboratories, Inc.). Nontransformed NIH 3T3 cells and v-Abl-transformed NIH 3T3 cells were as we described (72, 92). A431 cells (CRL-1555) were obtained from ATCC (Manassas, VA). A variety of other reagents were purchased commercially: FBS (JRH Biosciences, Lenexa, KS); murine TNF and IL-6 (PharMingen, San Diego, CA), bovine insulin (Sigma, St. Louis, MO); human EGF, human PDGF, and human bFGF (Upstate Biotechnology, Inc.); prestained protein markers (Life Technologies, Inc., Gaithersburg, MD); Slow-Fade antifade reagent (Molecular Probes, Inc., Eugene, OR).

Hybridoma Production
A mAb to phosphocaveolin-1 was generated by immunization of Balb/c female mice with a tyrosine-phosphorylated caveolin-1 peptide [residues 9–18; SEGHL(pY)TVPI]. This sequence is absolutely conserved in human, cow, mouse, rat, and dog caveolin-1. Mice showing the highest titer of immunoreactivity in RSV-transformed cells were used to create fusions with myeloma cells using standard protocols (99). Positive hybridomas were cloned twice by limiting dilution and injected into mice to produce ascites fluid. IgGs were purified by affinity chromatography on protein A-Sepharose.

Tyrosine-Phosphorylated GST-Caveolin-1
The GST-Cav-1 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101) fusion protein (in the vector pGEX-4T-1) was as we described (17, 23, 32). This fusion protein was expressed into two different Escherichia coli strains [either BL21 (DE3) for nonphosphorylated caveolin-1 or TKB1 for tyrosine- phosphorylated caveolin-1]. The TKB1 strain is a derivative of BL21 (DE3), which harbors a plasmid-encoded IPTG-inducible tyrosine kinase gene [the Elk receptor tyrosine kinase domain; Stratagene, La Jolla, CA (100)].

Cell Culture
NIH 3T3 cells were grown in DMEM supplemented with glutamine, antibiotics (penicillin and streptomycin), and 10% donor bovine calf serum (92). Cos-7, A431, and 293Tcells were grown in DMEM supplemented with glutamine, antibiotics (penicillin and streptomycin), and 10% FCS (17). 3T3-L1 fibroblasts were propagated and differentiated into adipocytes as described (101).

Transient Expression in Cos-7 Cells
Constructs encoding untagged caveolin-1 and C-terminally Myc epitope-tagged forms of caveolin-1, -2, or -3, were described by us previously (7, 17, 18, 20). A construct encoding untagged caveolin-1 (Y14A) was generated via PCR mutagenesis using oligonucleotides. These constructs ( 400 ng) were transiently transfected into Cos-7 cells alone or in combination with Src using the Effectene transfection reagent (QIAGEN, Chatsworth, CA), as per the manufacturer’s instructions. Forty-eight hours post transfection, cells were scraped into boiling sample buffer. Recombinant expression was analyzed by SDS-PAGE (15% acrylamide)/Western-blotting. Untagged caveolin-1 was detected using anticaveolin-1 IgG [mAb 2297; recognizes residues 61–71 of caveolin-1 (17)], which allows the detection of both Cav-1 (residues 1–178) and Cav-1ß (residues 32–178) isoforms. Epitope-tagged forms of caveolin-1, caveolin-2, and caveolin-3 were detected using the mAb 9E10, which recognizes the Myc epitope (EQKLISEEDLN).

IP/Western of Caveolin-1 (WT) and Caveolin-1 (Y14A)
Cos-7 cells were transiently cotransfected with caveolin-1 (WT) or caveolin-1 (Y14A), alone or in combination with c-Src (WT). Thirty-six hours post transfection, the cells were processed for immunoprecipitation using protein A-Sepharose CL-4B (Amersham Pharmacia Biotech, Arlington Heights, IL). Briefly, cells were lysed in immunoprecipitation (IP) buffer containing 10 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 60 mM octyl-glucoside with phosphatase inhibitor (50 mM NaF, 30 mM Na-pyrophosphate, 100 µM Na-orthovanadate), and protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). Lysates were precleared by addition of 50 µl of 1:1 slurry of protein A-Sepharose in TNET buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, 1% Triton X-100) containing 1 mg/ml BSA. After 30 min at 4 C, samples were centrifuged for 5 sec at 15,000 g. The resulting supernatants were transferred to fresh tubes, and 50 µl of protein A-Sepharose were added together with anticaveolin-1 IgG (H-97; Santa Cruz Biotechnology, Inc.; a rabbit pAb directed against the C-terminal domain of caveolin-1). Samples were then incubated for an additional 3 h at 4 C. immunoprecipitates were washed five times with IP buffer, and samples were separated by 12.5% SDS-PAGE and transferred to nitrocellulose. Blots were then probed with a well characterized mAb directed against phosphotyrosine (PY20; BD Transduction Laboratories, Inc.).

Immunofluorescence Microscopy
All reactions were performed at room temperature. Cos-7 cells or NIH 3T3 fibroblasts were briefly washed three times with PBS and fixed for 45 min in PBS containing 3% paraformaldehyde. Fixed cells were rinsed with PBS and treated with 25 mM NH₄Cl in PBS for 10 min to quench free aldehyde groups. Cells were then permeabilized with 0.1% Triton X-100 for 10 min at room temperature and washed four times with PBS for 10 min each time. Cells were then successively incubated with PBS/2% BSA containing 1) a 1:400 dilution of antiphosphocaveolin-1 IgG (mAb 56) and anticaveolin-1 IgG (pAb N-20; directed against caveolin-1 residues 2–21) and 2) LRSC-conjugated goat antimouse antibody (5 µg/ml) and FITC (fluorescein isothiocyanate)-conjugated donkey antirabbit antibody (5 µg/ml). The first incubation was 30 min, while primary and secondary antibody reactions were 60 min each. Cells were washed three time with PBS between incubations. Slides were mounted with Slow-Fade antifade reagent and observed under a MR600 confocal fluorescence microscope (Bio-Rad Laboratories, Inc., Hercules, CA). Note that both antiphosphocaveolin-1 (mAb 56; directed against residues ⁹ SEGHL(pY)TVPI¹⁸) and anticaveolin-1 (pAb N-20; directed against residues ² SGGKYVDSEGHLYTVPIR-EQ ²¹) recognize a very similar epitope present in Cav-1 (residues 1–178), but which is lacking in Cav-1ß (residues 32–178). Double-labeling experiments were also carried out in a similar fashion with antiphosphotyrosine IgG (pAb and mAb) and anti-paxillin IgG.

Charge-Coupled Device (CCD) Imaging and Deconvolution
Using a Ix80 microscope (Olympus Corp., Lake Success, NY) with a 60x Plan Neofluar objective and a Photometrics cooled CCD camera with a 35-mm shutter, the images were acquired and processed using IP Laboratory on a Power Mac 8500. For each sample, three to five two-dimensional images were acquired, deconvolved, and then combined into one two-dimensional image.

Immunoblotting with Antiphosphocaveolin-1 IgG
Cells were lysed in boiling sample buffer (67). Samples were then collected and boiled for a total of 5 min. Samples were homogenized using a 26 g needle and a 1-ml syringe. Cellular proteins were resolved by SDS-PAGE (13% acrylamide) and transferred to nitrocellulose membranes (0.2 µm). Blots were incubated for 2 h in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2% Tween 20) containing 2% powdered skim milk and 1% BSA. After three washes with TBST, membranes were incubated for 2 h with the primary antibody ( 1,000-fold diluted in TBST) and for 1 h with horseradish peroxidase-conjugated goat antirabbit/mouse IgG ( 5,000-fold diluted). Proteins were detected using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech).

Triton Insolubility
NIH 3T3 cells were washed twice with PBS and lysed for 30 min at 4 C in a buffer containing 10 mM Tris, pH 8.0, 0.15 M NaCl, 5 mM EDTA, and 1% Triton X-100 (49). Samples were centrifuged at 14,000 rpm for 10 min at 4 C. Pellet (I, insoluble) and supernatant (S, soluble) fractions were resolved by SDS-PAGE (12.5% acrylamide) and analyzed by immunoblotting.

Velocity Gradient Centrifugation
NIH 3T3 cells were dissociated in MES-buffered saline containing 60 mM octyl-glucoside. Solubilized material was loaded atop a 5–40% linear sucrose gradient and centrifuged at 50,000 rpm for 10 h in a SW 60 rotor (Beckman Instruments, Inc./Hybritech ) (7, 18, 20, 23). Gradient fractions were collected from above and subjected to immunoblot analysis. Molecular mass standards for velocity gradient centrifugation were as we described previously (7, 18, 20, 23).

Preparation of Caveolae-Enriched Membrane Fractions
NIH 3T3 cells were washed with PBS and lysed with 2 ml of Mes-buffered saline (MBS, 25 mM Mes, pH 6.5, 0.15 M NaCl) containing 1% (vol/vol) Triton X-100 (6, 17, 27, 28, 32, 41, 50, 60, 102, 103, 104). Homogenization was carried out with 10 strokes of a loose-fitting Dounce homogenizer. The homogenate was adjusted to 40% sucrose by the addition of 2 ml of 80% sucrose prepared in MBS and placed at the bottom of an ultracentrifuge tube. A 5–30% linear sucrose gradient was formed above the homogenate and centrifuged at 39,000 rpm for 16–20 h in a SW41 rotor (Beckman Instruments, Inc./Hybritech). A light scattering band confined to the 15–20% sucrose region was observed that contained caveolin-1 but excluded most other cellular proteins. From the top of each gradient, 1 ml gradient fractions were collected to yield a total of 12 fractions. An equal volume from each gradient fraction was separated by SDS-PAGE and subjected to immunoblot analysis.

In Vitro Phosphorylation
Caveolin-rich domains were purified from murine lung tissue, as described previously (17, 28). Caveolin-rich membrane domains ( 5 µg) were then resuspended in 20 µl of kinase buffer (20 mM HEPES, pH 7.4, 1 mM MgCl₂, 1 mM MnCl₂) and the reaction was initiated by addition of 1 mM ATP. After 10 min at room temperature, the reaction was halted by addition of 20 µl of 2x SDS-sample buffer and boiling for 2 min. To evaluate the effects of kinase inhibitors, samples were preincubated for 30 min at 4 C in the presence of a given inhibitor before initiation of the reaction. Tyrosine kinase inhibitors (tyrophostin AG1478, tyrophostin A9, and PP2) were purchased from Calbiochem (La Jolla, CA), dissolved in dimethylsulfoxide (DMSO), and used at a final concentration of 5 µM.

Detection of SH2 Domain Proteins That Bind Tyrosine-Phosphorylated Caveolin-1
Purified nonphosphorylated and tyrosine-phosphorylated GST-Cav-1 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101) fusion proteins were immobilized on glutathione-agarose beads and incubated with lysates from normal NIH 3T3 cells. These lysates were generated with IP buffer containing a battery of phosphatase and protease inhibitors [10 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 60 mM octyl-glucoside, 50 mM NaF, 30 mM Na-pyrophosphate, 100 µM Na- orthovanadate, pepstatin A (1 µg/ml), and 1 tab of complete protease inhibitor cocktail (Roche Molecular Biochemicals)]. After incubation rotating overnight at 4 C, the beads were washed with lysis buffer (5x), separated by 12.5% SDS-PAGE, and transferred to nitrocellulose membranes. Bound proteins were visualized by immunoblotting with a panel of antibodies directed against 20 known SH2 domain- containing proteins (see Table 2). These antibodies were purchased from BD Transduction Laboratories, Inc.

Grb7 Expression
A mammalian expression vector encoding murine Grb7 was generated as follows. Briefly, an expressed sequence tag (EST) containing the full-length murine Grb7 cDNA (Genbank Accession no. AI746340; clone ID no. 2065197) was purchased from Research Genetics, Inc. (Huntsville, AL). Using PCR-assisted subcloning, the full-length wild-type Grb7 cDNA was cloned into the multiple cloning site (SalI/XbaI) of the CMV-based pCB7 vector. For immunoprecipitation, an affinity-purified rabbit pAb directed against the C terminus of murine Grb7 (C-20; Santa Cruz Biotechnology, Inc.) was used. For Western blot analysis, a rabbit pAb directed against the N-terminal region of Grb7 was used (BD Transduction Laboratories, Inc.).

Grb7/Caveolin-1 Coimmunoprecipitation Experiments
Cos-7 cells were cotransfected with the cDNAs encoding caveolin-1, Grb7, and c-Src WT. Forty-eight hours post transfection, the cells were lysed in IP buffer containing phosphatase and protease inhibitors (detailed above) and subjected to immunoprecipitation with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech). Briefly, lysates were first precleared by addition of a 50 µl of 1:1 slurry of protein A-Sepharose in TNET buffer (defined above) containing 1 mg/ml BSA. After 30 min of preclearing at 4 C, samples were centrifuged for 5 sec at 15,000 x g, and the supernatants were transferred to fresh tubes. Then, 50 µl of protein A-Sepharose were added together with an irrelevant IgG (2 µg/ml) or Grb7 pAb IgG (2 µg/ml; C-20; Santa Cruz Biotechnology, Inc.). After incubation for 3 h at 4 C, the immunoprecipitates were washed three times with IP buffer, and the samples were separated by 12.5% SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with mAb 2297 to detect total caveolin-1 or mAb 56 to detect tyrosine 14- phosphorylated caveolin-1. Similar experiments were carried out comparing the coimmunoprecipitation of WT and Y14A forms of caveolin-1.

Grb7/Caveolin-1 Peptide Competition
Cos-7 cells were transiently transfected with the cDNAs encoding caveolin-1, Grb7, and c-Src WT. Forty-eight hours post transfection, cells were lysed in IP buffer containing phosphatase and protease inhibitors. After preclearing, antibodies directed against Grb7 (2 µg/ml; C-20; Santa Cruz Biotechnology, Inc.) and a given caveolin-1-derived competing peptide (200 µg/ml; PY14, Y14, or PY100) were added to the cell lysates. After incubation for 3 h at 4 C, the immunoprecipitates were washed three times with IP buffer, and the samples were separated by 12.5% SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with mAb cl 56 to detect tyrosine 14-phosphorylated caveolin-1.

Caveolin-1 Peptide/Grb7 Pull-Down Assay
Cos-7 cells were transiently transfected with the cDNA encoding Grb7. Forty-eight hours post transfection, cells were lysed in 1 ml of IP buffer containing phosphatase and protease inhibitors. After preclearing, biotinylated caveolin-1-derived phosphopeptides (either PY14 or PY100) were added prebound to streptavidin agarose beads. After incubation for 3 h at 4 C, the beads were washed three times with IP buffer, and the samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with a rabbit pAb directed against the N terminus of Grb7 (BD Transduction Laboratories, Inc.). To generate streptavidin beads containing prebound biotinylated peptides, the beads (50 µl) were incubated for 3 h with a 1 ml solution containing approximately 15 µg/ml of peptide dissolved in TNET buffer.

Focus Formation Assay
293T cells were transiently transfected with c-Src, Grb7, and Cav-1 (WT or Y14A), individually or in combination, using the calcium phosphate precipitation method. Three 60-mm dishes were used for each experimental condition. Forty-eight hours post transfection, the plates were examined under the light microscope using low magnification (4x or 6x), and the number of foci per plate was counted. Only foci greater than 1 mm in diameter were scored. Experimental values represent the average numbers of foci per plate for each experimental condition; error bars represent the observed SD.

Cell Migration Assay
A 48-well microchemotaxis chamber (NeuroProbe, Cabin John, MD) was used to study the chemotactic response to EGF as described previously (74), following the manufacturer’s instructions. Briefly, 293T cells were transiently transfected with c-Src, Grb7, and Cav-1 (WT or Y14A), individually or in combination, using the calcium phosphate precipitation method. Thirty-six hours post transfection, cells were incubated in serum-free media for 3 h before loading. Nucleopore filters (8-µm pore size) were coated with rat tail collagen I in PBS (27 µg/ml) for 2 h. After the lower wells of the chamber were filled with DMEM containing EGF (100 ng/ml), the filter was laid over the lower chamber and the whole chamber was assembled. An equal number of cells (2 x 10⁴/well) were suspended in DMEM and loaded into the upper wells and allowed to migrate for 3 h in a 37 C humidified incubator. Six wells were used for each experimental condition. At the end of the experiment, the cells that did not migrate across the membrane were scraped, and the cells that migrated were fixed in 3.7% formaldehyde in PBS and stained in hematoxylin overnight. The number of migrating cells was counted under a microscope.

	ACKNOWLEDGMENTS

 
We thank the members of the Pestell, Scherer, and Lisanti laboratories for helpful discussions, Dr. Anne Lane Schubert for help with CCD imaging and deconvolution, and Dr. Feng Dong for help with caveolae purification.

	FOOTNOTES

 
Address requests for reprints to: Dr. Michael P. Lisanti, Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Room 202 Golding Building, New York, New York 10461. E-mail: lisanti{at}aecom.yu.edu

This work was supported by grants from the National Institutes of Health, the Muscular Dystrophy Association (MDA), and the Susan G. Komen Breast Cancer Foundation (to M.P.L.). R.G.P. was supported in part by NIH Grants R01-CA-70897, R01-CA-75503, and P50-HL-56399; R.G.P. is a recipient of the Irma T. Hirschl award and an award from the Susan G. Komen Breast Cancer Foundation. H.L. was supported by an NIH Training Program grant. P.E.S. was supported by a grant from Pfizer, Inc., a pilot grant from the AECOM Diabetes Research and Training Center, and by a research grant from the American Diabetes Association.

Received for publication June 5, 2000. Revision received July 18, 2000. Accepted for publication August 8, 2000.

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TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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				H. Lee, S. E. Woodman, J. A. Engelman, D. Volonte', F. Galbiati, H. L. Kaufman, D. M. Lublin, and M. P. Lisanti Palmitoylation of Caveolin-1 at a Single Site (Cys-156) Controls Its Coupling to the c-Src Tyrosine Kinase. TARGETING OF DUALLY ACYLATED MOLECULES (GPI-LINKED, TRANSMEMBRANE, OR CYTOPLASMIC) TO CAVEOLAE EFFECTIVELY UNCOUPLES c-Src AND CAVEOLIN-1 (TYR-14) J. Biol. Chem., September 7, 2001; 276(37): 35150 - 35158. [Abstract] [Full Text] [PDF]

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