Soltoff S P, Cantley L C
Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02115, USA.
J Biol Chem. 1996 Jan 5;271(1):563-7. doi: 10.1074/jbc.271.1.563.
Although epidermal growth factor (EGF) activates phosphoinositide (PI) 3-kinase activity in a number of types of cells or cell lines, in most cases that we have investigated the p85 regulatory subunit of PI 3-kinase does not appear to bind directly to the EGF receptor. Previously we demonstrated that EGF-dependent activation of PI 3-kinase activity in A431 cells is accompanied by the binding of p85 to ErbB3, an EGF receptor homologue. However, this mechanism did not explain the large activation of PI 3-kinase activity that was found in PC12 and A549 cells, which possess little or no ErbB3. Here we provide evidence that the p120cbl proto-oncoprotein is an intracellular adapter protein that associates with PI 3-kinase and thus is involved in the EGF-dependent activation of this enzyme in these two cell lines. Using an anti-p120cbl antibody, we immunoprecipitated the EGF receptor from PC12 cells and PI 3-kinase activity from PC12 and A549 cells in an EGF-dependent fashion. Treatment of PC12 cells with nerve growth factor or insulin stimulated large increases in PI 3-kinase activity that was immunoprecipitated using anti-Tyr(P) antibody but not using anti-p120cbl antibody. In EGF-treated PC12 cells, the tyrosine phosphorylation of p120cbl displayed similar kinetics to the activation of PI 3-kinase as measured by both in vivo lipid production and lipid kinase assays conducted using anti-p120cbl and anti-Tyr(P) immunoprecipitates. The use of glutathione S-transferase fusion proteins of various domains of p85 demonstrated that p120cbl associated with both the SH2 and SH3 domains of p85. p120cbl was also present in A431 cells and offers an additional pathway by which EGF can activate PI 3-kinase in these cells.
尽管表皮生长因子（EGF）可在多种类型的细胞或细胞系中激活磷酸肌醇（PI）3激酶活性，但在我们研究的大多数情况下，PI 3激酶的p85调节亚基似乎并不直接与EGF受体结合。此前我们证明，A431细胞中EGF依赖性的PI 3激酶活性激活伴随着p85与ErbB3（一种EGF受体同源物）的结合。然而，这种机制无法解释在几乎不表达或不表达ErbB3的PC12和A549细胞中发现的PI 3激酶活性的大量激活。在此我们提供证据表明，p120cbl原癌蛋白是一种细胞内衔接蛋白，它与PI 3激酶相关联，因此在这两种细胞系中参与了EGF依赖性的该酶激活过程。使用抗p120cbl抗体，我们以EGF依赖性方式从PC12细胞中免疫沉淀出EGF受体，并从PC12和A549细胞中免疫沉淀出PI 3激酶活性。用神经生长因子或胰岛素处理PC12细胞可刺激PI 3激酶活性大幅增加，该活性可使用抗酪氨酸磷酸化（Tyr(P)）抗体而非抗p120cbl抗体进行免疫沉淀。在经EGF处理的PC12细胞中，p120cbl的酪氨酸磷酸化表现出与PI 3激酶激活相似的动力学，这通过体内脂质生成以及使用抗p120cbl和抗Tyr(P)免疫沉淀物进行的脂质激酶测定来衡量。使用p85不同结构域的谷胱甘肽S-转移酶融合蛋白证明，p120cbl与p85的SH2和SH3结构域均相关联。p120cbl也存在于A431细胞中，并为EGF在这些细胞中激活PI 3激酶提供了一条额外途径。
