Ezrin/moesin in motile Walker 256 carcinosarcoma cells: signal-dependent relocalization and role in migration.
Rat Walker 256 carcinosarcoma cells spontaneously develop front-tail polarity and migrate in the absence of added stimuli. Constitutive activation of phosphatidylinositol-3 kinase (PI 3-kinase), Rac, Rho and Rho kinase are essential for these processes. Ezrin and moesin are putative targets of these signaling pathways leading to spontaneous migration. To test this hypothesis, we used specific siRNA probes that resulted in a downregulation of ezrin and moesin by about 70% and in a similar reduction in the fraction of migrating cells. Spontaneous polarization however was not affected, indicating a more subtle role of ezrin and moesin in migration. We provide furthermore evidence that endogenous ezrin and moesin colocalize with F-actin at the contracted tail of polarized cells, similar to ectopically expressed green fluorescent protein-tagged ezrin. Our results suggest that myosin light chain and ezrin are markers of front and tail, respectively, even in the absence of morphological polarization. We further show that endogenous ezrin and moesin are phosphorylated and that activities of PI-3 kinase, Rho and Rac, but not of Rho-kinase, are required for this C-terminal phosphorylation. Activation of protein kinase C in contrast suppressed phosphorylation of ezrin and moesin. Inhibition of ezrin phosphorylation prevented its membrane association.
Rossy
Jérémie
J
Department of Pathology, University of Bern, Murtenstr. 31, CH-3010 Bern, Switzerland.
Gutjahr
Marc C
MC
Blaser
Nelsy
N
Schlicht
Dominique
D
Niggli
Verena
V
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D002279:Carcinoma 256, Walker; D002465:Cell Movement; D016764:Cell Polarity; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000502:physiology*; D015536:Down-Regulation; D004789:Enzyme Activation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology; D008840:Microfilament Proteins / Q000378:metabolism / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering; D051381:Rats; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine; D014162:Transfection; D000077191:Wortmannin; D020830:rac1 GTP-Binding Protein / Q000235:genetics; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases; D000099149:Ezrin
D000730:Androstadienes; D002273:Carcinogens; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D013755:Tetradecanoylphorbol Acetate; D013912:Threonine; D000077191:Wortmannin; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D054460:rho-Associated Kinases; D000099149:Ezrin; C071058:moesin; C071776:radixin