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J. Biol. Chem., Vol. 281, Issue 15, 9852-9858, April 14, 2006

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Pctaire1 Phosphorylates N-Ethylmaleimide-sensitive Fusion Protein

IMPLICATIONS IN THE REGULATION OF ITS HEXAMERIZATION AND EXOCYTOSIS^*

From the Department of Biochemistry, Biotechnology Research Institute, and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China

Pctaire1, a member of the cyclin-dependent kinase (Cdk)-related family, has recently been shown to be phosphorylated and regulated by Cdk5/p35. Although Pctaire1 is expressed in both neuronal and non-neuronal cells, its precise functions remain elusive. We performed a yeast two-hybrid screen to identify proteins that interact with Pctaire1. N-Ethylmaleimide-sensitive fusion protein (NSF), a crucial factor in vesicular transport and membrane fusion, was identified as one of the Pctaire1 interacting proteins. We demonstrate that the D2 domain of NSF, which is required for the oligomerization of NSF subunits, binds directly to and is phosphorylated by Pctaire1 on serine 569. Mutation of this phosphorylation site on NSF (S569A) augments its ability to oligomerize. Moreover, inhibition of Pctaire1 activity by transfecting its kinase-dead (KD) mutant into COS-7 cells enhances the self-association of NSF. Interestingly, Pctaire1 associates with NSF and synaptic vesicle-associated proteins in adult rat brain. To investigate whether Pctaire1 phosphorylation of NSF is involved in 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. Interestingly, expression of either Pctaire1-KD or NSF-S569A in PC12 cells significantly increases high K⁺-stimulated growth hormone release. Taken together, our findings provide the first demonstration that Pctaire1 phosphorylation of NSF regulates the ability of NSF to oligomerize, implicating an unexpected role of this kinase in modulating exocytosis. These findings open a new avenue of research in studying the functional roles of Pctaire1 in the nervous system.

Received for publication, December 19, 2005 , and in revised form, January 1, 2006.

^* This work was supported in part by the Research Grants Council of Hong Kong (HKUST 6091/01M and HKUST 6130/03M), the Area of Excellence Scheme of the University Grants Committee (AoE/B-15-01), and the Hong Kong Jockey Club. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of the Croucher Foundation Senior Research Fellowship. To whom correspondence should be addressed: Dept. of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China. Tel.: 852-2358-7304; Fax: 852-2358-2765; E-mail: BOIP{at}UST.HK.

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