||Pctaire1, a member of the cyclin-dependent kinase (Cdk)-related kinase family, has recently been identified as a substrate of Cdk5, where Cdk5-mediated phosphorylation enhances Pctaire1 activity. Although Pctaire1 is expressed in both neuronal and non-neuronal cells, its precise functions remain elusive. To examine the potential functions of Pctaire 1, we performed a yeast two-hybrid screen and mass spectrometry to identify proteins that interact with Pctaire1. Interestingly, Pctaire1 was observed to interact with several synaptic vesicle-associated proteins including syntaxin 1, synaptotagmin and synapsin I. In particular, N-ethylmaleimide sensitive fusion protein (NSF), a crucial factor in vesicular transport and membrane fusion, was also identified as one of the Pctaire1-interacting proteins. Further studies revealed that the D2 domain of NSF, which is required for the oligomerization of NSF subunits, bind directly to, and was phosphorylated by Pctaire1 at serine-569. Mutation of this phosphorylation site on NSF (S569A) augmented its ability to oligomerize. Moreover, inhibition of Pctaire1 activity by transfecting its kinase-dead (KD) mutant into COS-7 cells enhanced the self-association of NSF. These observations collectivley suggest that phosphorylation of NSF by Pctaire1 may negatively regulate oligomerization of NSF, thereby affecting NSF-mediated membrane fusion events in exocytosis. To investigate whether NSF phosphorylation by Pctaire1 is involved in the regulation of Ca2+-dependent exocytosis, we examined the effect of expressing Pctaire1 or NSF phosphorylation mutants on the regulated secretion of growth hormone from PC12 cells. Importantly, expression of either Pctaire1-KD or NSF-S569A in PC12 cells significantly increased high K+-stimulated growth hormone release, indicating that Pctaire1 phosphorylation of NSF suppressed high K+-stimulated growth hormone release. Taken together, our findings provide the first demonstration that phosphorylation of NSF by Pctaire1 regulates its oligomerization, which may result in the suppression of Ca2+-dependent exocytosis. More importantly, the concomitant identification of multiple synaptic vesicle-associated proteins as Pctaire1-interating protein implicates a functional role of Pctaire1 in the regulation of synaptic vesicle physiology. Further studies on the potential interaction of these synaptic vesicle-associated proteins with Pctaire1 will help unravel the functional roles of Pctaire1 in the nervous systems.