Phosphorylation of PDZ1 domain attenuates NHERF-1 binding to cellular targets.
NHERF-1 (Na(+)-H(+) exchanger regulatory factor 1, also known as EBP50 ezrin-binding protein of 50 kDa) is a phosphoprotein that assembles multiprotein complexes via two PDZ domains and a C-terminal ezrin-binding domain. Current work utilized metabolic labeling in cultured cells expressing wild type GFP-NHERF-1 to define the physiological importance of NHERF-1 phosphorylation. Treatment of cells with phosphatase inhibitors calyculin A and okadaic acid enhanced NHERF-1 phosphorylation and inhibited its dimerization. Eliminating C-terminal serines abolished the modulation of NHERF-1 dimerization by phosphatase inhibitors and identified the phosphorylation of the PDZ1 domain that attenuated its binding to physiological targets, including beta(2)-adrenergic receptor, platelet-derived growth factor receptor, cystic fibrosis transmembrane conductance regulator, and sodium-phosphate cotransporter type IIa. The major covalent modification of PDZ1 was mapped to serine 77. Confocal microscopy of cultured cells suggested key roles for PDZ1 and ERM-binding domain in localizing NHERF-1 at the cell surface. The substitution S77A eliminated PDZ1 phosphorylation and increased NHERF-1 localization at the cell periphery. In contrast, S77D reduced NHERF-1 colocalization with cortical actin cytoskeleton. These data suggested that serine 77 phosphorylation played key role in modulating NHERF-1 association with plasma membrane targets and identified a novel mechanism by which PDZ1 phosphorylation may transduce hormonal signals to regulate the function of membrane proteins in epithelial tissues.
Voltz
James W
JW
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
Brush
Matthew
M
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
Sikes
Suzanne
S
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
Steplock
Deborah
D
Department of Medicine, University of Maryland School of Medicine, Department of Veterans Affairs, Baltimore, Maryland 21201.
Weinman
Edward J
EJ
Department of Medicine, University of Maryland School of Medicine, Department of Veterans Affairs, Baltimore, Maryland 21201.
Shenolikar
Shirish
S
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710. Electronic address: Sheno001@mc.duke.edu.
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D019281:Dimerization; D006801:Humans; D008387:Marine Toxins; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D019319:Okadaic Acid / Q000737:chemistry; D010080:Oxazoles / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000378:metabolism
D008387:Marine Toxins; D010080:Oxazoles; D010750:Phosphoproteins; D018343:Receptors, Adrenergic, beta-2; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D019319:Okadaic Acid; C059041:calyculin A