Constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase C

• Published in: Molecular biology of the cell 2000-10, Vol.11 (10), p.3453-3467
• Main Authors: Amyere, M, Payrastre, B, Krause, U, Van Der Smissen, P, Veithen, A, Courtoy, P J
• Format: Article
• Language: English
• Subjects: Animals; Avian Sarcoma Viruses - genetics; Cattle; Cell Line; Cell Transformation, Neoplastic; Enzyme Activation; Genes, src; Genetic Vectors; Horseradish Peroxidase - pharmacokinetics; Kinetics; Microscopy, Electron, Scanning; Organelles - physiology; Organelles - ultrastructure; Phosphatidylinositol 3-Kinases - genetics; Phosphatidylinositol 3-Kinases - metabolism; Pinocytosis - physiology; Protein Subunits; Rats; Recombinant Proteins - metabolism; Sequence Deletion; Transfection; Type C Phospholipases - metabolism
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.11.10.3453

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 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 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.