title: Differential role of SNAP-25 phosphorylation by protein kinases A and C in the regulation of SNARE complex formation and exocytosis in PC12 cells.
abstract: The final step of regulated exocytosis, membrane fusion, is mediated by formation of the SNARE complex by syntaxin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and VAMP (vesicle-associated membrane protein). Phosphorylation of SNARE and accessory proteins contributes to regulation of exocytosis. We previously identified residues of SNAP-25 phosphorylated by protein kinase A (PKA) and PKC. However, the physiological role of SNAP-25 phosphorylation in exocytosis, in particular with regard to SNARE complex formation, has remained elusive. SNARE complex formation by purified recombinant SNAP-25, syntaxin-1, and VAMP-2 in vitro was inhibited or promoted as a result of the phosphorylation at Thr(138) by PKA or at Ser(187) by PKC, respectively. SNARE complex formation in intact PC12 cells was similarly inhibited by forskolin (activator of PKA) and promoted by phorbol 12-myristate 13-acetate (PMA, activator of PKC). Noradrenaline secretion from PC12 cells induced by a high K(+) concentration was enhanced by forskolin or PMA. Stable depletion of SNAP-25 inhibited high-K(+)-induced noradrenaline secretion. Forced expression of WT SNAP-25 restored the secretory response of the SNAP-25-depleted cells to high-K(+), and this response was enhanced by forskolin or PMA. Expression of the nonphosphorylatable T138A or S187A mutants of SNAP-25 similarly rescued the secretory response to high-K(+), but the augmentation of this response by forskolin was more pronounced in the cells expressing SNAP-25 (T138A) than in those expressing SNAP-25 (WT), whereas that by PMA was less pronounced in those expressing SNAP-25 (S187A). Our results thus suggest that SNAP-25 phosphorylation by PKA or PKC contributes differentially to the control of exocytosis in PC12 cells by regulating SNARE complex formation.
authors.affiliation: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Jing
authors.initials: J
authors.lastname: Gao
authors.affiliation: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; Section of Orthodontics, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Makiko
authors.initials: M
authors.lastname: Hirata
authors.affiliation: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Mizokami
authors.affiliation: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Section of Orthodontics, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Takahashi
authors.affiliation: Division of Applied Pharmacology, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takeuchi
authors.affiliation: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: hirata1@dent.kyushu-u.ac.jp.
authors.forename: Masato
authors.initials: M
authors.lastname: Hirata
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005089:Exocytosis; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000378:metabolism*; D050827:Syntaxin 1 / Q000378:metabolism
chemical_list: D000596:Amino Acids; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D009638:Norepinephrine
keywords: Exocytosis; Protein kinase; SNARE