Direct interaction between the cytoplasmic tail of ADAM 12 and the Src homology 3 domain of p85α activates phosphatidylinositol 3-kinase in C2C12 cells
Direct interaction between the cytoplasmic tail of ADAM 12 and the Src homology 3 domain of p85α activates phosphatidylinositol 3-kinase in C2C12 cells
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DOI：
10.1074/jbc.m101162200
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发表时间：
2001-07-06
影响因子：
4.8
通讯作者：
Zolkiewska, A
中科院分区：
文献类型：
--
作者：
Kang, Q;Cao, Y;Zolkiewska, A
ADAM 12是跨膜金属蛋白酶-去整合素的ADAM家族的成员，先前已经涉及骨骼肌成肌细胞的分化。在本研究中，我们证明了小鼠ADAM 12的胞质尾区在体外和体内与磷脂酰肌醇（PI）3-激酶的p85 α调节亚基的Src同源3结构域相互作用。通过定点突变，我们鉴定了ADAM 12中三个p85 α结合位点，包括位于氨基酸825-828，833-836，和884-887，使用绿色荧光蛋白（GFP）-普列克底物蛋白同源（PH）结构域融合蛋白作为PI 3-激酶脂质产物的探针，我们进一步证明了C2 C12细胞中ADAM 12的表达导致cFP-PH易位至质膜。这表明跨膜ADAM 12通过为p85 α的Src同源性3结构域提供对接位点，通过介导PI 3-激酶募集到膜来激活PI 3-激酶。由于PI 3-激酶对成肌细胞的终末分化至关重要，并且由于ADAM 12的表达在分化过程开始时上调，因此ADAM 12介导的激活可能构成成肌细胞分化期间PI I-激酶的调节机制之一。
ADAM 12, a member of the ADAM family of transmembrane metalloprotease-disintegrins, has been implicated previously in the differentiation of skeletal myoblasts. In the present study, we show that the cytoplasmic tail of mouse ADAM 12 interacts in vitro and in vivo with the Src homology 3 domain of the p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, By site-directed mutagenesis, we have identified three p85 alpha -binding sites in ADAM 12 involving PXXP motifs located at amino acids 825-828, 833-836, and 884-887, Using green fluorescent protein (GFP)-pleckstrin homology (PH) domain fusion protein as a probe for PI 3-kinase lipid products, we have further demonstrated that expression of ADAM 12 in C2C12 cells resulted in translocation of c;FP-PH to the plasma membrane. This suggests that transmembrane ADAM 12, by providing docking sites for the Src homology 3 domain of p85 alpha, activates PI 3-kinase by mediating its recruitment to the membrane. Because PI 3-kinase is critical for terminal differentiation of myoblasts, and because expression of ADAM 12 is up-regulated at the onset of the differentiation process, ADAM 12-mediated activation may constitute one of the regulatory mechanisms for PI I-kinase during myoblast differentiation.