Src tyrosine kinases, G(alpha) subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin - Caveolin binding negatively regulates the auto-activation of Src tyrosine kinases
Src tyrosine kinases, G(alpha) subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin - Caveolin binding negatively regulates the auto-activation of Src tyrosine kinases
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DOI：
10.1074/jbc.271.46.29182
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发表时间：
1996-11-15
影响因子：
4.8
通讯作者：
Lisanti, MP
中科院分区：
文献类型：
--
作者：
Li, SW;Couet, J;Lisanti, MP
小窝是大多数细胞类型中存在的质膜特殊性，可窝蛋白是22 kDa积分膜蛋白，是小窝膜的主要结构和调节成分。先前的研究表明，小窝蛋白与脂质修饰的信号分子共纯化，包括G（alpha）亚基，H-RAS C-SRC和其他相关的SRC家族酪氨酸激酶。另外，已经表明，小窝蛋白直接与G（alpha）亚基和H-RAS相互作用，优先识别这些分子的不活跃构象。但是，尚不清楚小窝蛋白是直接或间接与SRC家族酪氨酸激酶相互作用。在这里，我们研究了小窝蛋白与SRC家族酪氨酸激酶的结构和功能相互作用。小窝蛋白被重组表示为谷胱甘肽S-转移酶融合。使用已建立的体外结合测定法，我们发现小窝蛋白与野生型SRC（C-SRC）相互作用，但与突变激活的SRC（V-SRC）形成稳定的复合物。因此，小窝蛋白似乎更喜欢SRC的非活性构象。缺失诱变表明，小窝蛋白的SRC相互作用结构域位于残基82-101（可爱蛋白的胞质膜透明区域）中。小窝蛋白肽衍生出HOM（残基82-101）在功能上抑制了相关的SRC家族酪氨酸激酶的纯化重组C-SRC酪氨酸激酶和FYN的自动激活。我们进一步分析了可窝蛋白在293T细胞中瞬时共表达全长的小窝蛋白和C-SRC酪氨酸激酶，进一步分析了小窝蛋白对体内C-SRC活性的影响。与小窝蛋白共表达通过通过免疫复合激酶测定法测量的C-SRC的酪氨酸激酶活性。因此，似乎可以通过小窝蛋白Caveolin残基82-101在结构和功能上与野生型C-SRC相互作用。除了与SRC家族激酶相互作用外，该胞质小窝蛋白结构域（残基82-101）具有以下独特特征。首先，需要形成小窝蛋白的多价同型植物。其次，它与G蛋白α-亚基相互作用并下调其GTPase活性。第三，它与野生型H-RAS结合。第四，它是膜蛋白质，表明它可能与其他潜在的蛋白质蛋白质相互作用有关。因此，我们称这20个氨基酸的可爱可窝残基在小窝蛋白脚手架结构域中。
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 G(alpha) subunits, H-ras c-Src, and other related Src family tyrosine kinases. In addition, it has been shown that caveolin interacts directly with G(alpha) 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 hom 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.