Protein kinase C-dependent phosphorylation of synaptosome-associated protein of 25 kDa at Ser187 potentiates vesicle recruitment.
Activation of protein kinase C (PKC) constitutes a key event in the upregulation of secretory strength in neurons and neurosecretory cells during extensive stimulation, presumably by speeding up vesicle supply. However, the molecular targets and their mode of action remain elusive. We studied the only PKC-dependent phosphorylation site in the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Ser(187), in synaptosome-associated protein of 25 kDa (SNAP-25). This phosphorylation site is located within the negatively charged C-terminal end of SNAP-25, which has been shown to be of critical importance in calcium-triggered exocytosis. We combined mutational studies that used overexpression in chromaffin cells with capacitance measurements and flash photolysis of caged calcium, allowing for high time resolution during both the stimulation and measurement of exocytosis. Overexpression of mutants simulating the phosphorylated form of Ser(187) accelerated vesicle recruitment after the emptying of the releasable vesicle pools. Overexpression of mutants simulating the nonphosphorylated form, or block of PKC, impaired the refilling of the vesicle pools to similar extents. Biochemical studies verified the phosphorylation of a subpopulation of SNAP-25 after elevation of intracellular calcium concentrations. Some of the mutations led to a moderately decreased fast exocytotic burst component, which did not seem to be associated with the phosphorylation state of SNAP-25. Thus the C terminus of SNAP-25 plays a role for both fast exocytosis triggering and vesicle recruitment, and the latter process is regulated by PKC-dependent phosphorylation.
Nagy
Gábor
G
Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Matti
Ulf
U
Nehring
Ralf B
RB
Binz
Thomas
T
Rettig
Jens
J
Neher
Erwin
E
Sørensen
Jakob B
JB
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003198:Computer Simulation; D025802:Electric Capacitance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000187:drug effects; D010782:Photolysis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000378:metabolism*; D013329:Structure-Activity Relationship; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins
D000906:Antibodies; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D002118:Calcium