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The Journal of Neuroscience, November 1, 2002, 22(21):9278-9286
Protein Kinase C-Dependent Phosphorylation of
Synaptosome-Associated Protein of 25 kDa at
Ser187 Potentiates Vesicle Recruitment
Gábor
Nagy1,
Ulf
Matti2,
Ralf B.
Nehring1,
Thomas
Binz2,
Jens
Rettig1,
Erwin
Neher1, and
Jakob B.
Sørensen1
1 Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany, and 2 Institute for Physiological
Chemistry, Medical University Hannover, 30625 Hannover, Germany
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, Ser187, 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 Ser187 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.
Key words:
chromaffin cell; exocytosis; membrane capacitance; protein kinase C; SNARE proteins; SNAP-25
Copyright © 2002 Society for Neuroscience 0270-6474/02/22219278-09$05.00/0
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