Abstract
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.
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Change history
07 May 2012
This article has been corrected since Advance Online Publication and a Erratum is also printed in this issue
Abbreviations
- EBP50:
-
ERM binding phosphoprotein 50
- ERM:
-
ezrin/radixin/moesin
- GDI:
-
guanine nucleotide dissociation inhibitor
- GEF:
-
guanine nucleotide exchange factors
- GST:
-
glutathione S-transferase
- HGF:
-
hepatocyte growth factor
- PDZ:
-
postsynaptic density 95/discs large/zona occludens-1
- PKC:
-
protein kinase C
- PMA:
-
Phorbol-12-myristate-13-acetate
- PP2A:
-
protein phosphatase 2A
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Acknowledgements
This study is supported by National Science Council Grants (NSC 97-2320-B-002-048-MY3, and 99-3112-B-002-020) and National Taiwan University Hospital Grants (aNTUH-99P21-1) to T. S. Jou. We thank Professor W James Nelson (Stanford University) for English editing of this manuscript.
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Chen, JY., Lin, YY. & Jou, TS. Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis. Cell Death Differ 19, 1027–1037 (2012). https://doi.org/10.1038/cdd.2012.4
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DOI: https://doi.org/10.1038/cdd.2012.4
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