Phosphorylation of SNAP-23 in Activated Human Platelets

János Polgár, William S. Lane, Sul Hee Chung, Aiilyan K. Houng, Guy Reed

Research output: Contribution to journalArticle

65 Citations (Scopus)

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 α, 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 Ser 161, 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 phos. phorylation affect syntaxin 4 interactions, we hypothesize that SNAP-23 phosphorylation may be important for modulating SNARE-complex interactions during membrane trafficking and fusion.

Original languageEnglish (US)
Pages (from-to)44369-44376
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number45
DOIs
StatePublished - Nov 7 2003

Fingerprint

Phosphorylation
Platelets
Protein Kinase C
Blood Platelets
SNARE Proteins
Phosphopeptides
Qa-SNARE Proteins
Chemical activation
Thrombin
Membrane Fusion
Kinetics
Secretory Pathway
Exocytosis
Serine
Mass spectrometry
Brain
Mass Spectrometry
Proteins
Fusion reactions
Genes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Polgár, J., Lane, W. S., Chung, S. H., Houng, A. K., & Reed, G. (2003). Phosphorylation of SNAP-23 in Activated Human Platelets. Journal of Biological Chemistry, 278(45), 44369-44376. https://doi.org/10.1074/jbc.M307864200

Phosphorylation of SNAP-23 in Activated Human Platelets. / Polgár, János; Lane, William S.; Chung, Sul Hee; Houng, Aiilyan K.; Reed, Guy.

In: Journal of Biological Chemistry, Vol. 278, No. 45, 07.11.2003, p. 44369-44376.

Research output: Contribution to journalArticle

Polgár, J, Lane, WS, Chung, SH, Houng, AK & Reed, G 2003, 'Phosphorylation of SNAP-23 in Activated Human Platelets', Journal of Biological Chemistry, vol. 278, no. 45, pp. 44369-44376. https://doi.org/10.1074/jbc.M307864200
Polgár J, Lane WS, Chung SH, Houng AK, Reed G. Phosphorylation of SNAP-23 in Activated Human Platelets. Journal of Biological Chemistry. 2003 Nov 7;278(45):44369-44376. https://doi.org/10.1074/jbc.M307864200
Polgár, János ; Lane, William S. ; Chung, Sul Hee ; Houng, Aiilyan K. ; Reed, Guy. / Phosphorylation of SNAP-23 in Activated Human Platelets. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 45. pp. 44369-44376.
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