Src tyrosine kinases, Galpha subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin. Caveolin binding negatively regulates the auto-activation of Src tyrosine kinases

Abstract

Caveolae are plasma membrane specializations present in most cell types. Caveolin, a 22-kDa integral membrane protein, is a principal structural and regulatory component of caveolae membranes. Previous studies have demonstrated that caveolin co-purifies with lipid modified signaling molecules, including Galpha subunits, H-Ras, c-Src, and other related Src family tyrosine kinases. In addition, it has been shown that caveolin interacts directly with Galpha subunits and H-Ras, preferentially recognizing the inactive conformation of these molecules. However, it is not known whether caveolin interacts directly or indirectly with Src family tyrosine kinases. Here, we examine the structural and functional interaction of caveolin with Src family tyrosine kinases. Caveolin was recombinantly expressed as a glutathione S-transferase fusion. Using an established in vitro binding assay, we find that caveolin interacts with wild-type Src (c-Src) but does not form a stable complex with mutationally activated Src (v-Src). Thus, it appears that caveolin prefers the inactive conformation of Src. Deletion mutagenesis indicates that the Src-interacting domain of caveolin is located within residues 82-101, a cytosolic membrane-proximal region of caveolin. A caveolin peptide derived from this region (residues 82-101) functionally suppressed the auto-activation of purified recombinant c-Src tyrosine kinase and Fyn, a related Src family tyrosine kinase. We further analyzed the effect of caveolin on c-Src activity in vivo by transiently co-expressing full-length caveolin and c-Src tyrosine kinase in 293T cells. Co-expression with caveolin dramatically suppressed the tyrosine kinase activity of c-Src as measured via an immune complex kinase assay. Thus, it appears that caveolin structurally and functionally interacts with wild-type c-Src via caveolin residues 82-101. Besides interacting with Src family kinases, this cytosolic caveolin domain (residues 82-101) has the following unique features. First, it is required to form multivalent homo-oligomers of caveolin. Second, it interacts with G-protein alpha-subunits and down-regulates their GTPase activity. Third, it binds to wild-type H-Ras. Fourth, it is membrane-proximal, suggesting that it may be involved in other potential protein-protein interactions. Thus, we have termed this 20-amino acid stretch of caveolin residues the caveolin scaffolding domain.

FIG. 1. Examining the interaction of caveolin with c-Src and v-Src tyrosine kinases.

A, recombinant expression of c-Src and v-Src in insect Sf21 cells using baculovirus-based expression vectors (see “Experimental Procedures”). After infection, insect cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane. The expression of Src tyrosine kinases was monitored by immunoblot analysis with a mAb that recognizes the conserved extreme N-terminal of Src. This region is identical in c-Src and v-Src. Note that both c-Src and v-Src migrate at 60 kDa as indicated. B, GST-FL-Cav (bound to glutathione-agarose beads) was incubated with detergent extracts of insect cells recombinantly expressing c-Src or v-Src as shown in A (derived from two t25 flasks containing ~1 × 10⁶ cells each). GST-FL-Cav represents full-length caveolin (residues 1–178) expressed as a glutathione S-transferase (GST) fusion protein. After binding, extensive washing, and elution with reduced glutathione, bound Src was visualized by immunoblot analysis. Equivalent amounts of GST and GST-FL-Cav were used in these binding experiments. Note that c-Src specifically binds to GST-FL-Cav but not to GST alone. In striking contrast, v-Src failed to bind to either GST-FL-Cav or GST alone. These results with v-Src further demonstrate the specificity of the interaction between c-Src and caveolin.

FIG. 2.. Defining a region of caveolin that contains c-Src binding activity.

A, diagrammatic summary of each GST-caveolin fusion protein relative to a complete caveolin molecule. The numbers at end points reflect their exact amino acid position within caveolin. These fusion proteins correspond to the full-length caveolin protein (residues 1–178), the C-terminal caveolin domain (residues 135–178), and various portions of the N-terminal domain of caveolin (residues 1–21,1–41, 1–61, 1–81, or 61–101). These GST-caveolin fusion proteins were constructed and characterized as we described previously (24, 25, 27). B, molecular mapping of a caveolin region that interacts with c-Src. Using a panel of GST-caveolin fusion proteins enumerated in A, we systematically identified a 41-amino acid region of caveolin (residues 61–101) that is functionally sufficient to interact with c-Src. After binding, extensive washing, and elution with reduced glutathione, bound Src was visualized by immunoblot analysis as in Fig. 1B. Detergent extracts of insect cells recombinantly expressing c-Src were prepared from from a total of four t25 flasks containing ~1 × 10⁶ cells each. Equivalent amounts of GST and GST-caveolin fusion proteins were used in these binding experiments.

FIG. 3.. Effects of caveolin peptides on the auto-activation of c-Src tyrosine kinase.

Caveolin peptides, detailed in Table I, were examined for their effect on the autophosphorylation of purified recombinant c-Src kinase in vitro. The effects of peptide 1 (A), peptide 2 (B), and peptides 3 and 4 (C) are shown. Note that peptide 1 had no effect, whereas peptide 2 dose-dependently suppressed the auto-phosphorylation of c-Src. Peptide 2 encodes residues 82–101 of caveolin that contain caveolin binding activity as we have shown in Fig. 2B using GST-caveolin fusion proteins. Peptides 3 and 4 correspond to the N-terminal and C-terminal halves of peptide 2. Note that peptides 3 and 4 have no effect, indicating that the complete 82–101 region of caveolin (peptide 2) is required for inhibiting the auto-activation of c-Src. Quantitation of these experiments is provided in D. Cumulative data are shown as the means ± S.D. These experiments were performed at least three times independently in duplicate.

FIG. 4.. Effects of caveolin peptides on the auto-activation of Fyn, a closely related Src family tyrosine kinase.

Caveolin peptides were examined for their effect on the autophosphorylation of purified recombinant Fyn kinase in vitro. The effects of peptide 1 (A) and peptide 2 (B) are shown. Note that peptide 1 had no effect, whereas peptide 2 dose-dependently suppressed the auto-activation of Fyn. Quantitation of these experiments is provided in C. Cumulative data are shown as the means ± S.D. These experiments were performed at least three times independently in duplicate.

FIG. 5.. In vivo effect of the full-length caveolin molecule on the auto-activation of c-Src tyrosine kinase revealed by co-expression of caveolin and c-Src in 293T cells.

A, 293T cells (~1–2 × 10⁶ cells/10-cm dish) were transfected with c-Src alone or co-transfected with c-Src plus caveolin. 48 h post-transfection, cells were washed and collected in lysis buffer. Cell lysates were immunoprecipitated with anti-Src IgG bound to protein A-Sepharose. These immunoprecipitates were subjected to immunoblot analysis with a Src mAb probe (top panel), an immune complex kinase assay to detect Src auto-phosphorylation (middle panel), and immunoblot analysis with a caveolin mAb probe (bottom panel). Note that although both immunoprecipitates contain equivalent amounts of c-Src (top panel), co-expression with caveolin prevents the auto-phosphorylation of c-Src (middle panel). Also, caveolin co-immunoprecipitates with c-Src when using antibodies directed against c-Src (bottom panel). One 10-cm dish was used per immunoprecipitation. B, quantitation of A (middle panel). The auto-phosphorylation of c-Src tyrosine kinase is expressed in arbitrary units.

FIG. 6.. Localization of c-Src and caveolin within a single cell.

293T cells were co-transfected with c-Src and untagged mammalian caveolin. c-Src expression was detected with a mouse mAb that recognizes a conserved N-terminal epitope; caveolin expression was detected using rabbit polyclonal IgG directed against caveolin. Control experiments using singly transfected populations of cells confirmed the specificity of these antibodies; no cross-reaction was observed (not shown). Transfected cells expressing both c-Src and caveolin were selected for imaging by laser confocal fluorescence microscopy. Primary antibodies were detected using distinctly tagged fluorescent secondary antibodies: A, rhodamine-conjugated for c-Src; B, fluorescein-conjugated for caveolin; and C, superposition of the fluorescent images of A and B. Note that co-localization of c-Src and caveolin generates a yellow fluorescent image.

FIG. 7.. Effects of mutated caveolin peptides on the auto-activation of c-Src tyrosine kinase.

Wild-type and mutant caveolin peptides detailed in A were examined for their effect on the auto-phosphorylation of purified recombinant c-Src kinase in vitro. The effects of these peptides are shown in B. All peptides were added at a concentration of 1 μM. Note that both mutated caveolin peptides lacking tyrosine residues (Y→A and Y→F) were as effective or more effective than the wild-type (WT; peptide 2, residues 82–101) peptide sequence in suppressing the auto-phosphorylation of c-Src. The Y → F peptide was approximately twice as potent as the wild-type caveolin peptide.

FIG. 8.. Differential effect of peptides derived from caveolins-1, −2, and −3 on the auto-activation of c-Src tyrosine kinase.

Recently, two caveolin-related proteins (caveolin-2 and caveolin-3) have been identified and cloned; caveolin has been retermed caveolin-1. As a consequence, we also evaluated the effects of peptides derived from caveolins-2 and −3 that correspond to the 82–101 region in caveolin-1; the sequences of these peptides are detailed in A. All three peptides contain two conserved tyrosine residues (marked by arrows) and are extremely homologous. The effects of these peptides are shown in B. All peptides were added at a concentration of 1 μm. Note that only peptides derived from caveolins-1 and −3 exhibited inhibitory effects; the caveolin-2-derived peptide had no inhibitory effect.