Constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase C.
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.
Amyere
M
M
Cell Unit, Université Catholique de Louvain and Christian de Duve Institute of Cellular Pathology, 1200 Brussels, Belgium.
Payrastre
B
B
Krause
U
U
Van Der Smissen
P
P
Veithen
A
A
Courtoy
P J
PJ
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D004789:Enzyme Activation; D016391:Genes, src; D005822:Genetic Vectors; D006735:Horseradish Peroxidase / Q000493:pharmacokinetics; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D015388:Organelles / Q000502:physiology / Q000648:ultrastructure; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010873:Pinocytosis / Q000502:physiology*; D021122:Protein Subunits; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D021122:Protein Subunits; D011994:Recombinant Proteins; D006735:Horseradish Peroxidase; D019869:Phosphatidylinositol 3-Kinases; D010738:Type C Phospholipases