Nerve growth factor-induced phosphorylation of SNAP-25 in PC12 cells: a possible involvement in the regulation of SNAP-25 localization.
Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein essential for neurotransmitter release, is phosphorylated at Ser187 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 Ser187. 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 h 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 Ser187 to Ala abolished the NGF-dependent relocalization. A population of SNAP-25 in the plasma membrane was not increased by a point mutation at Ser187 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 PC12 cells.
Kataoka
M
M
Project Research Center, Mitsubishikasei Institute of Life Sciences, Tokyo, Japan.
Kuwahara
R
R
Iwasaki
S
S
Shoji-Kasai
Y
Y
Takahashi
M
M
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D008565:Membrane Proteins; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D050825:Synaptosomal-Associated Protein 25; D013997:Time Factors; D014018:Tissue Distribution; D015854:Up-Regulation
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D020932:Nerve Growth Factor