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.
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.
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Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479, USA. lisanti@wi.mit.edu
Couet
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Lisanti
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MP
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D051242:Caveolin 1; D022461:Caveolins; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D018631:ras Proteins / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; D019061:src-Family Kinases; D019204:GTP-Binding Proteins; D018631:ras Proteins