title: A novel role of Rho-kinase in the regulation of ligand-induced phosphorylated EGFR endocytosis via the early/late endocytic pathway in human fibrosarcoma cells.
abstract: The small GTPase RhoA and its downstream effectors, the Rho-associated kinase (Rho-kinase) family, are known to regulate cell morphology, motility, and tumor progression via the regulation of actin cytoskeleton rearrangement. In the present study, we evaluated the role of Rho-kinase in the intracellular endocytic trafficking of ligand-induced phosphorylated epidermal growth factor receptor (pEGFR). We investigated the time course of the internalization fate of EGF-induced pEGFR via the early/late endocytic pathway in human fibrosarcoma cell line HT1080 cells using Y-27632, a selective Rho-kinase inhibitor. We found, using confocal immunofluorescence microscopy and Western blot analysis, a large accumulation of pEGFR in the nuclei of HT1080 cells. In contrast, we observed decreased amounts of the pEGFR-positive staining in the nuclei along with an accumulation of cytosolic pEGFR staining when the cells were incubated for 15-30 min in the presence of Y-27632, implying that an aberrant endocytic trafficking mechanism of pEGFR occurs in HT1080 cells whereby pEGFR might be selectively translocated into the nucleus. Moreover, we demonstrated that after 15-min of stimulation with Texas Red-EGF, increasing numbers of pEGFR-positive staining that had colocalized with Texas Red-EGF-positive punctate staining were seen in the cytoplasm of HT1080 cells but after 30-min of stimulation, most of this staining had disappeared from the cytoplasm and a large accumulation of pEGFR-positive staining appeared in the nucleus. Thus, nuclear accumulation of pEGFR appears to occur in an EGF-dependent manner. In contrast, such nuclear pEGFR-positive staining was not seen in the Y-27632-treated cells. Furthermore, silencing of RhoA or Rho-kinases I/II by sequence specific siRNAs considerably inhibited the EGF-dependent nuclear accumulation of pEGFR. Collectively, these results provide the first evidence that Rho-kinase signaling pathway plays a suppressive role in the intracellular vesicle trafficking of pEGFR via the endocytic pathway and that an increased Rho-kinase activity leads to the attenuation of the normal endocytic vesicular traffic of pEGFR via the early/late endocytic pathway, instead causing pEGFR to be trafficked out of the endocytic vesicles into the nucleus.
authors.affiliation: Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. nishimur@bioc.phar.kyushu-u.ac.jp
authors.forename: Yukio
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Biborka
authors.initials: B
authors.lastname: Bereczky
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Yoshioka
authors.forename: Shun'ichiro
authors.initials: S
authors.lastname: Taniguchi
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Itoh
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005354:Fibrosarcoma / Q000201:enzymology* / Q000473:pathology*; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D008024:Ligands; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000577:Amides; D008024:Ligands; D011725:Pyridines; D034741:RNA, Small Interfering; C108830:Y 27632; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors; D054460:rho-Associated Kinases; D020830:rac1 GTP-Binding Protein