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- Title
Nerve Growth Factor-Induced Phosphorylation of SNAP-25 in PC12 Cells: A Possible Involvement in the Regulation of SNAP-25 Localization.
- Authors
Kataoka, Masakazu; Kuwahara, Reiko; Iwasaki, Satoshi; Shoji-Kasai, Yoko; Takahashi, Masami
- Abstract
Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein essential for neurotransmitter release, is phosphorylated at Ser[sup187] following activation of cellular protein kinase C by treatment with phorbol 12-myristate 13-acetate. However, it remains unclear whether neuronal activity or an endogenous ligand induces the phosphorylation of SNAP-25. Here we studied the phosphorylation of SNAP-25 in PC12 cells using a specific antibody for SNAP-25 phosphorylated at Ser[sup187]. A small fraction of SNAP-25 was phosphorylated when cells were grown in the absence of nerve growth factor (NGF). A brief treatment with NGF that was enough to activate the mitogen-activated protein kinase signal transduction pathway did not increase the phosphorylation of SNAP-25; however, phosphorylation was up-regulated after a prolonged incubation with NGF. Up-regulation was transitory, and maximum phosphorylation (a fourfold increase over basal phosphorylation) was achieved between 36 and 48 hours after the addition of NGF. Immunofluorescent microscopy showed that SNAP-25 was localized primarily in the plasma membrane, although a significant population was also present in the cytoplasm. Quantitative microfluorometry revealed that prolonged treatment with NGF resulted in a preferential localization of SNAP-25 in the plasma membrane. A mutational study using a fusion protein with green fluorescent protein as a tag indicated that the point mutation of Ser[sup187] to Ala abolished the NGF-dependent relocalization. A population of SNAP-25 in the plasma membrane was not increased by a point mutation at Ser[sup187] to Glu; however, it was increased by prolonged treatment with NGF, indicating that the SNAP-25 phosphorylation is essential, but not sufficient, for the NGF-induced relocation to the plasma membrane. Our results suggest a close temporal relationship between the up-regulation of SNAP-25 phosphorylation and its relocation, and NGF-induced differentiation of...
- Subjects
PROTEINS; PROTEIN kinase C; NERVE growth factor
- Publication
Journal of Neurochemistry, 2000, Vol 74, Issue 5, p2058
- ISSN
0022-3042
- Publication type
Article
- DOI
10.1046/j.1471-4159.2000.0742058.x