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J. Biol. Chem., Vol. 276, Issue 15, 12174-12181, April 13, 2001
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From the ¶ Neurology Service, Department of Veterans Affairs
Medical Center, Lexington, Kentucky 40511 and the Departments
of Critical to SNARE protein function in
neurotransmission are the accessory proteins, soluble
N-ethylmaleimide-sensitive factor (NSF) attachment protein
(SNAP), and NSF, that play a role in activation of the SNAREs for
membrane fusion. In this report, we demonstrate the
depolarization-induced, calcium-dependent phosphorylation of NSF in rat synaptosomes. Phosphorylation of NSF is coincident with
neurotransmitter release and requires an influx of external calcium.
Phosphoamino acid analysis of the radiolabeled NSF indicates a role for
a serine/threonine-specific kinase. Synaptosomal phosphorylation of NSF
is stimulated by phorbol esters and is inhibited by staurosporine, chelerythrine, bisindolylmaleimide I, calphostin C, and Ro31-8220 but
not the calmodulin kinase II inhibitor, Kn-93, suggesting a role for
protein kinase C (PKC). Indeed, NSF is phosphorylated by PKC in
vitro at Ser-237 of the catalytic D1 domain. Mutation of this
residue to glutamic acid or to alanine eliminates in vitro phosphorylation. Molecular modeling studies suggest that Ser-237 is
adjacent to an inter-subunit interface at a position where its
phosphorylation could affect NSF activity. Consistently, mutation of
Ser-237 to Glu, to mimic phosphorylation, results in a hexameric form
of NSF that does not bind to SNAP-SNARE complexes, whereas the
S237A mutant does form complex. These data suggest a negative regulatory role for PKC phosphorylation of NSF.
Phosphorylation of the N-Ethylmaleimide-sensitive
Factor Is Associated with Depolarization-dependent
Neurotransmitter Release from Synaptosomes*
,,, and
Biochemistry and § Neurology and
Pharmacology, University of Kentucky Medical Center,
Lexington, Kentucky 40536
*
This work was supported in part by the Veterans Affairs
Research Service, Department of Veterans Affairs, and by the University of Kentucky Medical Center grants (to J. T. S.) and National
Institutes of Health Grants NS21868 (to T. C. V.) and HL56652 (to
S. W. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 859-323-6702 (ext. 245); Fax: 859-323-1037; E-mail: jslevin@pop.uky.edu.
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