Phosphorylation of the N-ethylmaleimide-sensitive factor is associated with depolarization-dependent neurotransmitter release from synaptosomes.
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.
Neurology Service, Department of Veterans Affairs Medical Center, Lexington, Kentucky 40511, USA.
E A
EA
Matveeva
S W
SW
Whiteheart
T C
TC
Vanaman
J T
JT
Slevin
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D052066:N-Ethylmaleimide-Sensitive Proteins; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism* / Q000502:physiology; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D018377:Neurotransmitter Agents; D033921:Vesicular Transport Proteins; D012694:Serine; D011493:Protein Kinase C; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat