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J. Biol. Chem., Vol. 277, Issue 11, 8771-8774, March 15, 2002

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ACCELERATED PUBLICATION
A Phosphotyrosine-dependent Protein Interaction Screen Reveals a Role for Phosphorylation of Caveolin-1 on Tyrosine 14

RECRUITMENT OF C-TERMINAL Src KINASE^*

Haiming Cao, William E. Courchesne^§, and Cynthia Corley Mastick^¶

From the Departments of  Biochemistry and ^§ Microbiology, University of Nevada, Reno, Nevada 89557

Received for publication, November 16, 2001, and in revised form, January 10, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Caveolin-1 is a substrate for nonreceptor tyrosine kinases including Src, Fyn, and Abl. To investigate the function of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions. A cDNA library was screened using the N terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl. We identified two proteins in this screen that interact with caveolin-1 in a phosphorylation-dependent manner: tumor necrosis factor- receptor-associated factor 2 (TRAF2) and C-terminal Src kinase (Csk). TRAF2 bound to nonphosphorylated caveolin-1, but this association was increased 3-fold by phosphorylation. In contrast, association of Csk with caveolin-1 was completely dependent on phosphorylation of caveolin-1, both for fusion proteins in yeast (>35-fold difference in affinity) and for endogenous proteins in tissue culture cells. Our data suggest that phosphorylation of caveolin-1 leads to Csk translocation into caveolae. This may induce a feedback loop that leads to inactivation of the Src family kinases that are highly enriched in caveolae.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Caveolae are small, abundant plasma membrane invaginations that have been implicated in a number of cellular processes (1). Caveolae are formed at lipid rafts in membranes by their coat proteins, the caveolins (2, 3). Caveolin-1 gene-deficient mice show loss of caveolae, uncontrolled endothelial cell proliferation, and impaired nitric oxide and calcium signaling, indicating that caveolae and caveolin-1 play fundamental roles in organizing multiple signaling pathways (3). However, their exact roles in these processes remain unclear.

Caveolin-1 acts as a scaffolding protein and binds to signaling molecules through its 20-amino acid scaffolding domain (4). Caveolin-1 is also directly involved in signaling cascades as a substrate of both serine and tyrosine kinases. Caveolin-1 is phosphorylated on tyrosine 14 by Src, Fyn, and Abl in response to a number of stimuli, including insulin, angiotensin II, osmotic shock, and oxidative stress (5-7).¹ In fact, caveolin-1 is a preferred substrate for these tyrosine kinases in cells (9-12).¹ With the exception of sites within the activation loops of kinases themselves, tyrosine phosphorylation is used to bind to protein phosphotyrosine binding domains, particularly SH2² domains (13). This in turn leads to the activation of downstream signaling cascades. Therefore, phosphorylation of caveolin on tyrosine is likely to be an intermediate step in a signaling cascade occurring within caveolae. Caveolin-1 phosphorylated on tyrosine 14 would serve as a docking site for SH2 domain-containing proteins and would recruit proteins into caveolae to activate downstream signaling cascades.

To identify proteins that bind to phosphorylated caveolin-1 in an unbiased manner, we screened a 3T3-L1 adipocyte cDNA library for proteins that interact with phosphorylated caveolin-1 using a novel yeast dihybrid screen. We found that C-terminal Src kinase (Csk) bound to phosphorylated caveolin in yeast and in mammalian tissue culture cells. A possible role for Csk recruitment to caveolae in response to caveolin-1 phosphorylation is discussed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phosphotyrosine-dependent Dihybrid Protein Interaction Screen-- This protocol is a modification of the standard yeast dihybrid screen. The bait protein was the first 61 amino acids of caveolin-1 fused to the N terminus of the Gal4 DNA binding domain (DBD) expressed in the vector pG4BD (Dr. Robert M. Brazas, University of California, San Francisco, CA). This construct removes the caveolin scaffolding domain and a stretch of negatively charged amino acids that cause self-activation. The catalytic domain of the Abl tyrosine kinase (Dr. W. Todd Miller, State University of New York at Stony Brook, NY; Ref. 14) with a 5' nuclear localization sequence was integrated into the Ade2 locus of the yeast chromosome using a yeast integration vector YIpDCE1 (Dr. Robert Stearman, National Institutes of Health, Bethesda, MD; Ref. 15). A 3T3-L1 adipocyte cDNA library fused to the Gal4 activation domain (AD) was used to screen for caveolin-1-binding proteins or "prey" (Alan R. Saltiel, Life Sciences Institute, Ann Arbor, MI; Ref. 16). This library contains ~3 × 10⁶ independent clones. Yeast were initially selected for growth on His/3-amino-1,2,4-triazole (65 mM) plates. Of the 2 × 10⁷ transformants screened, 134 grew well on His plates (colonies >2 mm). Plasmid DNAs were isolated from each one. To identify false positives and self-activators, the plasmids were reintroduced into the original yeast strain or cells expressing no Gal4 DBD. The isolates were sorted into 53 independent clones by PCR amplification based on size of insert and restriction digestion pattern (using AluI). All 53 were se- quenced. Phosphorylation-dependent interactions were identified in counterscreens as those interactions that were lost in yeast strains expressing the caveolin-1 bait protein alone or expressing a caveolin-1 bait protein with tyrosine 14 changed to phenylalanine (caveolin-1/Y14F).

Immunoprecipitation-- NIH-3T3 or primary human fibroblast (HF) cells (150-mm plate; Ref. 12) were lysed in 1 ml of HNTG buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM Na₄P₂O₇, 100 mM NaF, 1 mM Na₃VO₄, 10 µg/ml aprotinin, 1 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride). HNTG buffer was used to preserve SH2 domain-phosphotyrosine interactions (17). Cell lysates were incubated on ice for 1 h to disrupt caveolae, and insoluble material was removed by centrifugation (10 min at 13,500 rpm in a microcentrifuge). This incubation was sufficient to solubilize >50% of the caveolin-1 in these cell lines. To eliminate nonspecific binding of caveolin-1, the lysates were added to 100 µl of packed beads (Sepharose CL-4B) and incubated on ice for 1 h, and the precleared beads were removed by centrifugation. This preclearing step was essential. For immunoprecipitation, 30 µl of protein A-Sepharose beads preloaded with or without 12 µg of polyclonal anti-Csk antibody were added to the precleared lysates (0.5 ml) and incubated at 4 °C for 3 h. The beads were pelleted by centrifugation. Immunoprecipitates were washed four times in HNTG buffer, eluted in SDS sample buffer, and analyzed by SDS-PAGE and Western blotting as described (12).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To identify proteins that bind to phosphorylated caveolin-1, we screened a cDNA library using a novel yeast dihybrid screen. We modified the Gal4-based yeast two-hybrid system to perform a phosphotyrosine-dependent dihybrid protein interaction screen (Fig. 1A). The principle of the phosphotyrosine-dependent dihybrid screen is to introduce a kinase (Abl) into the yeast two-hybrid system to phosphorylate the bait protein (caveolin-1) and then screen for phosphorylation-dependent protein interactions.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Phosphotyrosine-dependent yeast dihybrid protein interaction screen. A, screen: caveolin-1 (amino acids 1-61) fused to the Gal4 DBD was used as the "bait." The catalytic domain of Abl was expressed to phosphorylate caveolin-1. The prey was an adipocyte cDNA library fused to the Gal4 AD. Steps in the screen are as follows: (i) identify positive clones by selection and screening, (ii) counterscreen for phosphorylation-dependent interactions, and (iii) sequence to look for SH2 domains or phosphotyrosine binding domains. B, lysates from yeast expressing caveolin-1·Gal4 DBD, Abl, or both were analyzed by Western blotting using antibodies to caveolin-1 (Cav1), Gal4 DBD, Abl, and phosphocaveolin (PY14). C, lysates from yeast expressing Abl and caveolin-1·Gal4 DBD or caveolin-1/Y14F·Gal4 DBD (Cav1 Y14F) were analyzed.

Caveolin-1 is split into two cytoplasmic domains by a central hydrophobic membrane anchor. The only site of tyrosine phosphorylation on caveolin-1 is tyrosine 14. To mimic the native structure of caveolin-1 and prevent steric hindrance at the phosphorylation site, we expressed the N terminus of caveolin-1 as a fusion protein attached to the N terminus of the Gal4 DBD. Abl was chosen as the kinase because caveolin-1 is phosphorylated on tyrosine 14 by c-Abl in cells¹ and in vitro (12). The catalytic domain of c-Abl with a 5' nuclear localization signal was integrated into the yeast chromosome. The truncated kinase was used to avoid introduction of an SH2 domain-containing protein into the system (Abl domain structure: SH3, SH2, kinase domain, and tail). The expression of the caveolin-1·Gal4 DBD fusion protein and Abl and the phosphorylation of caveolin-1 in the yeast strains were confirmed by Western blotting (Fig. 1B). In the absence of Abl or when tyrosine 14 of caveolin-1 was changed to phenylalanine (Fig. 1C), no phosphocaveolin was detected. The positive and negative controls for the standard yeast two-hybrid screen still behaved as expected after the expression of Abl kinase in the yeast cells (data not shown).

A 3T3-L1 adipocyte cDNA library fused to the Gal4 AD was transformed into yeast expressing caveolin-1 and Abl, and transformants were selected for growth on His/3-amino-1,2,4-triazole plates. After elimination of false positives, self-activators, and duplicates, we identified eight "true positive" proteins that interact with the N terminus of caveolin-1. All but one of these were isolated multiple times.³ Of these eight proteins, three were known proteins, and five were unknown. The three known proteins were JAB1 (21 isolates), tumor necrosis factor- receptor-associated factor 2 (TRAF2) (10 isolates), and Csk (6 isolates). Full-length clones were isolated in all three cases, and at least two independent clones with different inserts were isolated for each one. Of these proteins, only Csk contains a known phosphotyrosine binding domain, an SH2 domain.

Based on the initial screen, positive proteins could interact with caveolin-1 in a phosphorylation-dependent or phosphorylation-independent manner. To counterscreen for phosphorylation-dependent interactions, all positive clones were transformed back into yeast that expressed caveolin-1 alone, caveolin-1 and Abl, or caveolin-1/Y14F and Abl (Fig. 2). Protein interactions were determined by liquid culture -galactosidase assays. The liquid culture assays were quantitative and efficiently identified phosphorylation-dependent interactions. Similar results were observed when the protein interactions were determined by selective growth or colony-lift filter -galactosidase assays (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Phosphorylation-dependent interaction of Csk with caveolin-1 in yeast cells. Csk (A), TRAF2 (B), and JAB1 (C) were transformed into yeast expressing caveolin-1 alone (column 1), caveolin-1 and Abl (column 2), or caveolin-1/Y14F and Abl (column 3). D, full-length c-Abl was used as a control SH2 domain-containing protein. -Galactosidase expression was determined by quantitative liquid culture assays CLONTECH Yeast Protocols Handbook. After subtracting values of the negative controls (Gal4 AD with caveolin-1·Gal4 DBD), the -galactosidase units were expressed as a percentage of the standard positive control (p53·Gal4 DBD and SV40 large T-antigen·Gal4 AD).

We found only one protein, Csk, whose interaction with caveolin-1 was completely dependent on phosphorylation (Fig. 2A). Csk activated the expression of reporter genes in cells that expressed both caveolin-1 and Abl but not in cells expressing caveolin-1 alone. The possibility that the catalytic domain of Abl acts as a bridge between Csk and caveolin-1 can be excluded because Csk did not interact with caveolin-1/Y14F despite the presence of Abl. The interaction of Csk with phosphocaveolin is moderate (23% of positive control) but significantly greater (35-45-fold) than the background levels detected with unphosphorylated caveolin-1 or caveolin-1/Y14F. To control for the specificity of the SH2 domain interaction, we also examined the interaction between full-length c-Abl and caveolin-1 in yeast cells (Fig. 2D). Full-length c-Abl did not show significant interaction with caveolin-1 or phosphocaveolin. This indicates that only specific SH2 domain-containing proteins will interact with a given tyrosine-phosphorylated bait protein in the dihybrid screen.

TRAF2 also interacted with caveolin-1 in a phosphorylation-dependent manner (Fig. 2B). While TRAF2 bound to nonphosphorylated caveolin-1 (50% of positive control), phosphorylation of caveolin-1 increased the binding of TRAF2 to caveolin-1 ~3-fold. TRAF2 also bound to caveolin-1/Y14F but at a lower affinity (25% of positive control). These data indicate that TRAF2 binding to caveolin-1 is mediated in part through tyrosine 14. The phosphorylation dependence of the interaction is surprising as TRAF2 does not have an identifiable phosphotyrosine binding domain. It has been shown that TRAF2 and caveolin-1 form a constitutive complex in mammalian cells (18). After ligand binding, the tumor necrosis factor- receptor redistributes in the membrane and binds to this complex. In contrast to Csk and TRAF2, the proteasome subunit JAB1 interacted with caveolin-1 in a phosphorylation-independent manner (Fig. 2C; ~45% of positive control in the three yeast strains).

To determine whether full-length caveolin-1 interacts with endogenous Csk, we next examined the interaction of these proteins in tissue culture cells (Fig. 3). The first cell line used was an NIH-3T3 cell line expressing a temperature-sensitive allele of v-Abl, ts-120. Caveolin-1 is phosphorylated on tyrosine in these cells as determined by both anti-phosphotyrosine and anti-phosphocaveolin Western blotting⁴ (Ref. 12; Fig. 3A, upper panel, Lysates). Csk was immunoprecipitated from v-Abl-expressing cells or control cells, and immunoprecipitates were analyzed by Western blotting. Phosphocaveolin was detected in Csk immunoprecipitates from the v-Abl-expressing cells but not from control cells (Fig. 3A, upper panel, IP: Csk). No phosphocaveolin was detected in mock immunoprecipitations (Fig. 3A, upper panel, NS). When a parallel blot was probed with anti-phosphotyrosine antibodies, we found that phosphocaveolin was one of two major phosphoproteins that bind to Csk in the v-Abl-expressing cells (Fig. 3A, lower panel, filled arrows). The specificity is quite remarkable considering the dramatic induction of total cellular tyrosine phosphorylation in v-Abl-expressing cells (Fig. 3A, lower panel, Lysates). The less intense 68-kDa phosphoprotein that coimmunoprecipitated with Csk comigrates with paxillin, a multidomain protein that localizes primarily to focal adhesions (Fig. 3A, lower panel, open arrow; data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Csk interacts with phosphocaveolin in mammalian cells. A, Csk was immunoprecipitated from cells expressing a temperature-sensitive v-Abl kinase (v-Abl) or control NIH-3T3 cells. Proteins bound to Csk antibody (IP) or control beads (NS) in the original cell lysates (Lys) or in the supernatants (Sup) were analyzed by Western blotting using antibodies to phosphocaveolin (PY14), Csk, and phosphotyrosine (pTyr). Filled arrows indicate antibody bands. B, Csk was immunoprecipitated from HF cells before (Control) or after induction of oxidative stress (H2O2, 5 mM, 15 min). Proteins were analyzed by anti-phosphocaveolin Western blotting. C, HF cells were infected with recombinant adenovirus expressing FLAG-tagged caveolin-1 (Cav) or caveolin-1/Y14F (CavY14F). Csk was immunoprecipitated from lysates prepared from infected cells before () or after (+) induction of oxidative stress. Cell lysates and Csk immunoprecipitates were analyzed by anti-FLAG or anti-phosphocaveolin Western blotting.

To further characterize the interaction between endogenous Csk and caveolin-1 without expression of v-Abl, we immunoprecipitated Csk from HF cells after oxidative stress.¹ Caveolin-1 was phosphorylated in cells treated with H₂O₂, while caveolin-1 phosphorylation was undetectable in control cells (Fig. 3B, Lysates). After oxidative stress, phosphocaveolin was detected in the Csk immunoprecipitates (Fig. 3B, IP: Csk).

While phosphocaveolin was detected in the Csk immunoprecipitates, available antibodies were not sensitive enough to detect unphosphorylated caveolin-1 in these samples. To overcome this limitation, we transiently expressed full-length FLAG epitope-tagged caveolin-1 or caveolin-1/Y14F in HF cells and looked for coimmunoprecipitation of the FLAG tag with Csk before and after oxidative stress. Overexpression of epitope-tagged caveolin-1 in HF cells was sufficient to induce significant phosphorylation of caveolin-1 in the lysates (Fig. 3C, upper panel, Lysates). However, the phosphorylation was dramatically increased after oxidative stress. Caveolin-1/Y14F was not phosphorylated on any tyrosine residues even after oxidative stress,¹ although phosphorylation of endogenous caveolin-1 was detected in the lysates of these cells (Fig. 3C, filled arrow).

In cells overexpressing caveolin-1, phosphocaveolin coimmunoprecipitated with Csk both before and after induction of oxidative stress (Fig. 3C, upper panel, IP: Csk). No phosphocaveolin was detected in Csk immunoprecipitates from cells expressing caveolin-1/Y14F under either condition. Caveolin-1, as detected by the FLAG epitope tag, could only be detected in Csk immunoprecipitates from cells expressing caveolin-1, not caveolin-1/Y14F, despite equal expression of both constructs (Fig. 3C, lower panel). These data indicate that Csk only binds to caveolin-1 after phosphorylation on tyrosine 14. Consistent with this, phosphocaveolin was enriched in the Csk immunoprecipitates relative to the whole cell lysates from untreated cells. These results support the idea that phosphorylation of caveolin on tyrosine 14 is a mechanism to control the interaction of Csk with caveolin-1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

What is the significance of the phosphorylation-dependent interaction of caveolin-1 and Csk? In cells, Csk negatively regulates the activities of Src family kinases by phosphorylating a conserved inhibitory tyrosine residue (527 in Src). However, the Src family kinases are localized to lipid rafts in the plasma membrane (including caveolae) via covalent lipid modifications, while Csk is not lipid modified and is largely free in the cytosol. The ability of Csk to relocate from the cytosol to the plasma membrane requires a functional SH2 domain on Csk but not the tyrosine phosphorylation sites on Src (19). Therefore, Csk must be recruited to its substrates at the plasma membrane via interaction with other tyrosine-phosphorylated proteins. Recently it has been shown in brain and cultured lymphocytes that Csk is recruited to lipid rafts in the plasma membrane through an SH2 domain interaction with a transmembrane protein called Cbp that is constitutively phosphorylated on tyrosine (20). The binding of Csk to tyrosine-phosphorylated Cbp activates Csk (21).

Our data indicate that caveolin-1 phosphorylation is another mechanism to recruit Csk to the plasma membrane. Csk binds specifically to phosphorylated caveolin-1 (Fig. 3). Caveolin-1 is one of only two phosphotyrosine proteins found to coimmunoprecipitate with Csk in the fibroblast cells used in our study (paxillin is the other). Despite the widespread distribution of Cbp, we did not detect a phosphotyrosine protein of the predicted size of Cbp in Csk immunoprecipitates from NIH-3T3 cells (Fig. 3). While caveolin-1 is expressed at very low levels in brain and lymphocytes, it is expressed at high levels in terminally differentiated cells such as endothelial cells, smooth muscle cells, and adipocytes. Therefore, caveolin-1 may be the major protein mediating Csk membrane localization in these cells.

Disruption of the Csk gene causes unregulated Src family kinase activity, increased phosphorylation of a number of substrate proteins involved in the regulation of the actin cytoskeleton, impaired stress fiber formation, and defects in cell-matrix adhesion and cell-cell adhesion (22). Interestingly a loss of caveolin-1 expression induces a number of these same effects, including increased Src kinase activity and disruption of cell-matrix adhesion (23). Several lines of evidence implicate caveolins in the regulation of the actin cytoskeleton. Disruption of caveolin-3 in mice and in humans leads to limb girdle muscular dystrophy, a disease caused by disruption of complexes between the actin cytoskeleton, the plasma membrane, and the extracellular matrix (2). Caveolin-1 at the cell surface often aligns along the underlying actin cytoskeleton, possibly through its interaction with filamin (24). In addition, phosphocaveolin is found in stabilized focal contacts in adherent cells (7), and caveolin-1 is phosphorylated in intact tissues where there are a large number of cell-cell and cell-matrix attachments (data not shown; Ref. 25).

The kinases known to phosphorylate caveolin-1, Abl, Src, and Fyn are also associated with focal adhesions. Upon integrin ligation activated Abl transiently associates with the forming focal contact (26), while Src is recruited and inactivated and remains in a stable association with these structures (27). The inactivation of Src in the focal contacts is due to phosphorylation of its inhibitory Csk site. Src activity is required for the turnover of cell-matrix contacts during cell migration and division in response to extracellular stimuli, not for focal contact formation (28, 29). Fyn is also involved in integrin signaling through a complex that requires caveolin-1 expression (8, 23).

Based on these observations we propose the following model. Activation of Abl or Src leads to the phosphorylation of caveolin-1 on tyrosine 14. This leads to the recruitment of Csk and phosphorylation and inactivation of Src family kinases that are highly enriched in caveolae. Caveolin phosphorylation would induce the formation of preassembled signaling complexes containing inactive Src family kinases at specific sites at the cell membrane that are primed for transient activation in response to extracellular stimuli. The caveolin-1/Csk/Src family kinase signaling complex may be involved in transmitting signals to the actin cytoskeleton (i.e. from integrins or oxidative stress) or transmitting signals from the actin cytoskeleton (i.e. from shear or osmotic stress).

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant DK56197.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, Mail Stop 330, University of Nevada, Reno, NV 89557. Tel.: 775-784-1155; Fax: 775-784-1419; E-mail: cmastick@med.unr.edu.

Published, JBC Papers in Press, January 22, 2002, DOI 10.1074/jbc.C100661200

¹ A. R. Sanguinetti and C. C. Mastick, manuscript submitted.

³ The library was screened at approximately 7-fold redundancy, therefore true positive clones are expected to be isolated multiple times.

⁴ Phosphocaveolin and a comigrating phosphotyrosine band coimmunoprecipitate with caveolin-1 from ts-120-expressing cells. Anti-phosphocaveolin blotting shows only bands that comigrate with caveolin-1 even in whole cell lysates.

	ABBREVIATIONS

The abbreviations used are: SH, Src homology; caveolin-1/Y14F, caveolin-1 with tyrosine 14 mutated to phenylalanine; Csk, C-terminal Src kinase; Gal4 AD and Gal4 DBD, the activation and DNA binding domains of Gal4, respectively; HF, human fibroblast; TRAF, tumor necrosis factor- receptor-associated factor.

	REFERENCES

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				H. Kogo, T. Aiba, and T. Fujimoto Cell Type-specific Occurrence of Caveolin-1{alpha} and -1{beta} in the Lung Caused by Expression of Distinct mRNAs J. Biol. Chem., June 11, 2004; 279(24): 25574 - 25581. [Abstract] [Full Text] [PDF]

				M. S. Duxbury, H. Ito, S. W. Ashley, and E. E. Whang CEACAM6 Cross-linking Induces Caveolin-1-dependent, Src-mediated Focal Adhesion Kinase Phosphorylation in BxPC3 Pancreatic Adenocarcinoma Cells J. Biol. Chem., May 28, 2004; 279(22): 23176 - 23182. [Abstract] [Full Text] [PDF]

				H. Wang, M. Haas, M. Liang, T. Cai, J. Tian, S. Li, and Z. Xie Ouabain Assembles Signaling Cascades through the Caveolar Na+/K+-ATPase J. Biol. Chem., April 23, 2004; 279(17): 17250 - 17259. [Abstract] [Full Text] [PDF]

				X.-Q. Wang, P. Sun, and A. S. Paller Ganglioside GM3 Blocks the Activation of Epidermal Growth Factor Receptor Induced by Integrin at Specific Tyrosine Sites J. Biol. Chem., December 5, 2003; 278(49): 48770 - 48778. [Abstract] [Full Text] [PDF]

				B. Nichols Caveosomes and endocytosis of lipid rafts J. Cell Sci., December 1, 2003; 116(23): 4707 - 4714. [Abstract] [Full Text] [PDF]