Phosphorylation of SNAP-23 in activated human platelets.
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.
Polgár
János
J
Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Lane
William S
WS
Chung
Sul-Hee
SH
Houng
Aiilyan K
AK
Reed
Guy L
GL
D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure; D002352:Carrier Proteins / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D003594:Cytoplasmic Granules / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011464:Epoprostenol / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000097:blood; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050600:SNARE Proteins; D012694:Serine / Q000097:blood; D050825:Synaptosomal-Associated Protein 25; D013917:Thrombin / Q000494:pharmacology; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011994:Recombinant Proteins; C100493:SNAP23 protein, human; C494208:SNAP25 protein, human; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D012694:Serine; D011464:Epoprostenol; D011493:Protein Kinase C; D013917:Thrombin