Phosphorylation of SNAP-23 in activated human platelets

J Biol Chem. 2003 Nov 7;278(45):44369-76. doi: 10.1074/jbc.M307864200. Epub 2003 Aug 20.

Abstract

Phosphorylation of SNARE proteins may provide a critical link between cell activation and secretory processes. Platelets contain all three members of the SNAP-23/25/29 gene family, but by comparison to brain tissue, SNAP-23 is the most highly enriched of these proteins in platelets. SNAP-23 function is required for exocytosis from platelet alpha, dense, and lysosomal granules. SNAP-23 was phosphorylated largely on serine residues in platelets activated with thrombin. Phosphorylation kinetics paralleled or preceded granule secretion. Inhibition studies suggested that SNAP-23 phosphorylation proceeds largely through a protein kinase C (PKC) mechanism and purified PKC directly phosphorylated recombinant (r-) SNAP-23 (up to 0.3 mol of phosphate/mol of protein). Five major tryptic phosphopeptides were identified in cellular SNAP-23 isolated from activated platelets; three phosphopeptides co-migrated with those identified in PKC-phosphorylated r-SNAP-23. In contrast, only one major phosphopeptide was identified when SNAP-23, engaged in a ternary SNARE complex, was phosphorylated by PKC. Ion trap mass spectrometry revealed that platelet SNAP-23 was phosphorylated at Ser23/Thr24 and Ser161, after cell activation by thrombin; these sites were also identified in PKC-phosphorylated r-SNAP-23. SNAP-23 mutants that mimic phosphorylation at Ser23/Thr24 inhibited syntaxin 4 interactions, whereas a phosphorylation mutant of Ser161 had only minor effects. Taken together these studies show that SNAP-23 is phosphorylated in platelets during cell activation through a PKC-related mechanism at two or more sites with kinetics that parallel or precede granule secretion. Because mutants that mimic SNAP-23 phosphorylation affect syntaxin 4 interactions, we hypothesize that SNAP-23 phosphorylation may be important for modulating SNARE-complex interactions during membrane trafficking and fusion.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Blood Platelets / metabolism*
  • Blood Platelets / ultrastructure
  • Carrier Proteins / blood*
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Cytoplasmic Granules / metabolism
  • Enzyme Inhibitors / pharmacology
  • Epoprostenol / pharmacology
  • Humans
  • Immunoblotting
  • Kinetics
  • Membrane Proteins / blood
  • Molecular Sequence Data
  • Mutagenesis
  • Nerve Tissue Proteins / blood
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Phosphorylation
  • Platelet Activation* / drug effects
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / blood
  • Qa-SNARE Proteins
  • Qb-SNARE Proteins
  • Qc-SNARE Proteins
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • SNARE Proteins
  • Serine / blood
  • Synaptosomal-Associated Protein 25
  • Thrombin / pharmacology
  • Vesicular Transport Proteins*

Substances

  • Carrier Proteins
  • Enzyme Inhibitors
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Peptide Fragments
  • Qa-SNARE Proteins
  • Qb-SNARE Proteins
  • Qc-SNARE Proteins
  • Recombinant Proteins
  • SNAP23 protein, human
  • SNAP25 protein, human
  • SNARE Proteins
  • Synaptosomal-Associated Protein 25
  • Vesicular Transport Proteins
  • Serine
  • Epoprostenol
  • Protein Kinase C
  • Thrombin