v-Src induces constitutive macropinocytosis in rat fibroblasts

The role of v-Src as regulator of fluid-phase pinocytosis was investigated in Rat-1 cells expressing a stable (Rat-1/BB16) or a thermosensitive (Rat-1/tsLA29) v-Src protein. In the second cell line, this protein is inactive when cells are cultured at 40 degrees C but recovers its tyrosine kinase activity upon transfer to 34 degrees C, resulting into a transformed phenotype. The rate of fluid-phase pinocytosis of the tracer horseradish peroxidase was 2-fold higher in v-Src-transformed fibroblasts (Rat-1/BB16, Rat-1/tsLA29 cultured at 34 degrees C) as compared to non-transformed cells (Rat-1, Rat-1/tsLA29 kept at 40 degrees C). In contrast, receptor-mediated endocytosis of transferrin was poorly affected, suggesting that structures distinct from clathrin-coated pits are involved in pinocytosis stimulation. By light and electron microscopy, transformed cells frequently contained large peroxidase-labeled pinocytic vesicles located near to membrane ruffles, demonstrating that stimulation of pinocytosis corresponds to induction of constitutive macropinocytosis. Stimulation of pinocytosis occurred more than 8 hours after transfer to the permissive temperature, whereas transfer to the non-permissive temperature partially reversed the stimulation within 2 hours. Protein synthesis inhibition for 6 hours abrogated pinocytosis stimulation in transformed cells, indicating that constitutive macropinocytosis induced by v-Src depends on continuous synthesis of a short-lived regulatory machinery.