LIM KINASE 1/COFILIN SIGNALING PATHWAY PLAYS A PIVOTAL ROLE IN REGULATING EGF RECEPTOR ENDOCYTOSIS IN INVASIVE HUMAN BREAST CANCER CELLS pp. 141-159
Authors: Yukio Nishimura, Kiyoko Yoshioka, Kazuyuki Itoh, Division of Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan, and others
Abstract: The small GTPase Rho and Rho-associated protein kinase (ROCK) signaling pathway has been demonstrated to be one of the major pathways involved in tumor invasion through reorganization of actin cytoskeleton. It was shown previously that an expression of constitutively active form of RhoA or of ROCK in rat hepatoma cells considerably promoted invasive ability of these cells in vitro and in vivo, and enhanced phosphorylation level of myosin light chain MLC20, thereby, indicating that Rho-ROCK pathway mediates tumor cell motility and invasion. ROCK can phosphorylate and activate LIM kinase 1 (LIMK1), which leads to phosphorylation of cofilin. LIMK 1 is known to play a critical role in actin cytoskeletal remodeling by linking the signal from the Rho family of small GTPases to the change in cofilin activity, thereby, indicating an important role for Rho-ROCK-LIMK1-cofilin signaling in tumor cell invasion through regulating actin dynamics. We reported previously that overexpression of LIMK1 resulted in a marked retardation of EGF receptor endocytosis in low-invasive human breast cancer cell MCF-7. Thereby, we postulate that LIMK 1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGF receptor in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining was accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomes after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGF receptor endocytosis, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15-30 min after internalization. Taken together, our novel findings imply that LIMK1-cofilin signaling indeed plays a pivotal role in the regulation of EGF receptor endocytosis via the early/late endocytic pathway in invasive tumor cells.