title: Protein kinase C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50.
abstract: Ezrin-Radixin-Moesin (ERM) binding phosphoprotein 50 (EBP50, a.k.a. NHERF-1) is a scaffold protein essential for the localization and coordinated activity of apical transporters, enzymes and receptors in epithelial cells. EBP50 acts via multiple protein binding interactions, including oligomerization through interactions of its PSD95-Dlg-ZO1 (PDZ) domains. EBP50 can be phosphorylated on multiple sites and phosphorylation of specific sites modulates the extent of oligomerization. The aim of the present study was to test the capacity of protein kinase C (PKC) to phosphorylate EBP50 and to regulate its oligomerization. In vitro experiments showed that the catalytic subunit of PKC directly phosphorylates EBP50. In HEK-293 cells transfected with rat EBP50 cDNA, a treatment with 12 myristate 13-acetate (PMA) induced a translocation of PKCalpha and beta isoforms to the membrane and increased 32P incorporation into EBP50. In co-transfection/co-precipitation studies, PMA treatment stimulated EBP50 oligomerization. Mass spectrometry analysis of full-length EBP50 and phosphorylation analyses of specific domains, and of mutated or truncated forms of EBP50, indicated that PKC-induced phosphorylation of EBP50 occurred on the Ser337/Ser338 residue within the carboxyl-tail domain of the protein. Truncation of Ser337/Ser338 also diminished PKC-induced oligomerization of EBP50. These results suggest the PKC signaling pathway can impact EBP50-dependent cellular functions by regulating EBP50 oligomerization.
authors.affiliation: Inserm U680, UPMC and Service de Biochimie et d'Hormonologie AP-HP, Hôpital Tenon, Faculté de Médecine Saint-Antoine, Paris, F-75012, France.
authors.forename: Laura
authors.initials: L
authors.lastname: Fouassier
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Nichols
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Gidey
authors.forename: Ryan R
authors.initials: RR
authors.lastname: McWilliams
authors.forename: Hélène
authors.initials: H
authors.lastname: Robin
authors.forename: Claire
authors.initials: C
authors.lastname: Finnigan
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Howell
authors.forename: Chantal
authors.initials: C
authors.lastname: Housset
authors.forename: R Brian
authors.initials: RB
authors.lastname: Doctor
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D013912:Threonine; D012694:Serine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate