Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis.
We demonstrated a protein kinase C (PKC)-dependent phosphorylation of canine ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) at serine 347/348 by site-directed mutagenesis and a phospho-specific antibody. Cell fractionation and confocal imaging revealed the relocation of EBP50 from the plasma membrane to cytosol that accompanied this phosphorylation event. Increased phosphorylation at these serine residues led to the dissociation of EBP50 from ezrin and β-PIX, which are two upstream regulators of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to hepatocyte growth factor-induced cell spreading and scattering, which is normally mediated by Rac1 activation. Detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation did not undergo apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function.
Graduate Institute of Molecular Medicine, National Taiwan University, No.7, Chung-Shan S. Road, Taipei 100, Taiwan. [corrected].
J-Y
JY
Chen
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Lin
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Jou
D000595:Amino Acid Sequence; D000818:Animals; D023102:Anoikis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004285:Dogs; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D054648:Protein Phosphatase 2 / Q000378:metabolism; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors; D012694:Serine / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D020662:Guanine Nucleotide Exchange Factors; D010750:Phosphoproteins; D011493:Protein Kinase C; D054648:Protein Phosphatase 2; D064067:Rho Guanine Nucleotide Exchange Factors; D012694:Serine; D020830:rac1 GTP-Binding Protein; D000099149:Ezrin; C576606:ARHGEF7 protein, human