A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells.
We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) 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-EGFR 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 were 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/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15-30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.
Nishimura
Yukio
Y
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan. nishimur@bioc.phar.kyushu-u.ac.jp
Yoshioka
Kiyoko
K
Bernard
Ora
O
Bereczky
Biborka
B
Itoh
Kazuyuki
K
D051339:Actin Depolymerizing Factors / Q000235:genetics / Q000502:physiology*; D001943:Breast Neoplasms / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D054461:Lim Kinases; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D011494:Protein Kinases / Q000502:physiology*; D021381:Protein Transport; D051116:Receptors, Scavenger / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured
D051339:Actin Depolymerizing Factors; D051907:Lysosomal Membrane Proteins; D051116:Receptors, Scavenger; C074299:SCARB2 protein, human; D011494:Protein Kinases; D066246:ErbB Receptors; C515892:LIMK1 protein, human; D054461:Lim Kinases; D002402:Cathepsin D