RRC ID 56041
Author Sakurai C, Itakura M, Kinoshita D, Arai S, Hashimoto H, Wada I, Hatsuzawa K.
Title Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor-mediated phagosome formation and maturation in macrophages.
Journal Mol Biol Cell
Abstract SNAP-23 is a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation-specific antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the nonphosphorylatable S95A or the phosphomimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D-overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Coexpression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ-activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.
Volume 29(13)
Pages 1753-1762
Published 2018-7-15
DOI 10.1091/mbc.E17-08-0523
PMID 29771640
PMC PMC6080709
MeSH Humans Interferon-gamma / pharmacology Lysosomes / drug effects Lysosomes / metabolism Macrophages / drug effects Macrophages / metabolism* Membrane Fusion / drug effects Models, Biological Mutant Proteins / metabolism Phagocytosis / drug effects Phagosomes / drug effects Phagosomes / metabolism* Phosphorylation / drug effects Phosphoserine / metabolism* Qb-SNARE Proteins / chemistry* Qb-SNARE Proteins / metabolism* Qc-SNARE Proteins / chemistry* Qc-SNARE Proteins / metabolism* Receptors, Fc / metabolism* Structure-Activity Relationship
IF 3.905
Times Cited 2
Resource
Human and Animal Cells J774.1