Phosphorylation of SNAP-23 regulates exocytosis from mast cells.
Regulated exocytosis is a process in which a physiological trigger initiates the translocation, docking, and fusion of secretory granules with the plasma membrane. A class of proteins termed SNAREs (including SNAP-23, syntaxins, and VAMPs) are known regulators of secretory granule/plasma membrane fusion events. We have investigated the molecular mechanisms of regulated exocytosis in mast cells and find that SNAP-23 is phosphorylated when rat basophilic leukemia mast cells are triggered to degranulate. The kinetics of SNAP-23 phosphorylation mirror the kinetics of exocytosis. We have identified amino acid residues Ser(95) and Ser(120) as the major phosphorylation sites in SNAP-23 in rodent mast cells. Quantitative analysis revealed that approximately 10% of SNAP-23 was phosphorylated when mast cell degranulation was induced. These same residues were phosphorylated when mouse platelet degranulation was induced with thrombin, demonstrating that phosphorylation of SNAP-23 Ser(95) and Ser(120) is not restricted to mast cells. Although triggering exocytosis did not alter the absolute amount of SNAP-23 bound to SNAREs, after stimulation essentially all of the SNAP-23 bound to the plasma membrane SNARE syntaxin 4 and the vesicle SNARE VAMP-2 was phosphorylated. Regulated exocytosis studies revealed that overexpression of SNAP-23 phosphorylation mutants inhibited exocytosis from rat basophilic leukemia mast cells, demonstrating that phosphorylation of SNAP-23 on Ser(120) and Ser(95) modulates regulated exocytosis by mast cells.
Hepp
Régine
R
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Puri
Niti
N
Hohenstein
Anita C
AC
Crawford
Garland L
GL
Whiteheart
Sidney W
SW
Roche
Paul A
PA
D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D002460:Cell Line; D005089:Exocytosis; D008407:Mast Cells / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D012694:Serine / Q000378:metabolism; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C498858:Snap23 protein, rat; D033921:Vesicular Transport Proteins; D012694:Serine