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Vol. 11, Issue 10, 3453-3467, October 2000

Constitutive Macropinocytosis in Oncogene-transformed Fibroblasts Depends on Sequential Permanent Activation of Phosphoinositide 3-Kinase and Phospholipase C

Mustapha Amyere,* Bernard Payrastre,dagger Ulrike Krause,Dagger Patrick Van Der Smissen,* Alex Veithen,* and Pierre J. Courtoy*§

 *Cell and  Dagger Hormone Units, Université Catholique de Louvain and Christian de Duve Institute of Cellular Pathology, 1200 Brussels, Belgium; and  dagger Institut National de la Santé et de la Recherche Médicale 326, 31059 Toulouse, France

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 p85alpha 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 3-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 p85alpha 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.

§ Corresponding author. E-mail address: courtoy{at}cell.ucl.ac.be.

Molecular Biology of the Cell
Vol. 11, 3453-3467, October 2000
Copyright © 2000 by The American Society for Cell Biology


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