PDZ interactions regulate rapid turnover of the scaffolding protein EBP50 in microvilli.
Scaffolding proteins containing PDZ (postsynaptic density 95/discs large/zonula occludens-1) domains are believed to provide relatively stable linkages between components of macromolecular complexes and in some cases to bridge to the actin cytoskeleton. The microvillar scaffolding protein EBP50 (ERM-binding phosphoprotein of 50 kD), consisting of two PDZ domains and an ezrin-binding site, retains specific proteins in microvilli and is necessary for microvillar biogenesis. Our analysis of the dynamics of microvillar proteins in vivo indicated that ezrin and microvillar membrane proteins had dynamics consistent with actin treadmilling and microvillar lifetimes. However, EBP50 was highly dynamic, turning over within seconds. EBP50 turnover was reduced by mutations that inactivate its PDZ domains and was enhanced by protein kinase C phosphorylation. Using a novel in vitro photoactivation fluorescence assay, the EBP50-ezrin interaction was shown to have a slow off-rate that was dramatically enhanced in a PDZ-regulated manner by addition of cell extract to near in vivo levels. Thus, the linking of relatively stable microvillar components can be mediated by surprisingly dynamic EBP50, a finding that may have important ramifications for other scaffolding proteins.
Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
Damien
D
Garbett
Anthony
A
Bretscher
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008871:Microvilli / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D054731:PDZ Domains; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099149:Ezrin
D000199:Actins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011493:Protein Kinase C; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C093376:sodium-hydrogen exchanger regulatory factor