Constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase C
Constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase C
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DOI：
10.1091/mbc.11.10.3453
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发表时间：
2000-10-01
影响因子：
3.3
通讯作者：
Courtoy, PJ
中科院分区：
文献类型：
--
作者：
Amyere, M;Payrastre, B;Courtoy, PJ
巨胞饮作用是由膜皱褶产生的板状伪足的关闭引起的，从而反映了皮层肌动蛋白的动态。通过v-Src或K-Ras转化Rat-1成纤维细胞和稳定转染显性阳性野生型磷酸肌醇3-激酶（PI 3 K）调节亚基p85 α表达，组成性导致应力纤维破坏，皮质肌动蛋白募集，广泛的皱褶和巨胞饮体形成，通过选择性加速液相内吞作用测量。这些变化与PI 3 K和磷脂酰肌醇特异性磷脂酶C（PI-PLC）的激活密切相关，分别通过原位和体外S-磷酸肌醇合成和1，4，5-三磷酸肌醇稳态水平进行检测;通过稳定转染v-Src转化的细胞进行显性负性截短的p85 α表达和通过PI 3 K和PI-PLC的药理学抑制剂，表明需要两种酶。尽管PI 3 K活化抵抗PI-PLC抑制，但PI-PLC活化被PI 3 K抑制剂和显性负转染消除，从而将PI-PLC置于PI 3 K下游。总之，这些数据表明，PI 3 K和PI-PLC的永久顺序激活是必要的，在癌基因转化的成纤维细胞中的肌动蛋白细胞骨架的戏剧性重组，导致组成性的皱褶和巨胞饮。
Macropinocytosis results from the closure of lamellipodia generated by membrane ruffling, thereby reflecting cortical actin dynamics. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection for expression of dominant-positive, wild-type phosphoinositide 3-kinase (PI3K) regulatory subunit p85 alpha constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and phosphatidylinositol-specific phospholipase C (PI-PLC), as assayed by S-phosphoinositide synthesis in situ and in vitro and inositol 1,4,5 trisphosphate steady-state levels, respectively; they were abolished by stable transfection of v-Src-transformed cells for dominant-negative truncated p85 alpha expression and by pharmacological inhibitors of PI3K and PI-PLC, indicating a requirement for both enzymes. Whereas PI3K activation resisted PI-PLC inhibition, PI-PLC activation was abolished by a PI3K inhibitor and dominant-negative transfection, thus placing PI-PLC downstream of PI3K. Together, these data suggest that permanent sequential activation of both PI3K and PI-PLC is necessary for the dramatic reorganization of the actin cytoskeleton in oncogene-transformed fibroblasts, resulting in constitutive ruffling and macropinocytosis.