Phosphorylation of 25-kDa Synaptosome-associated Protein

doi:10.1074/jbc.271.24.14548

Phosphorylation of 25-kDa Synaptosome-associated Protein

POSSIBLE INVOLVEMENT IN PROTEIN KINASE C-MEDIATED REGULATION OF NEUROTRANSMITTER RELEASE*

From the Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan and
^‡ Department of Veterinary Science, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan

^§ To whom correspondence should be addressed. Tel.: 81-427-24-6269; Fax: 81-427-24-6314; E-mail: masami{at}libra.ls.m-kagaku.co.jp

Abstract

Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells. Phorbol 12-myristate 13-acetate treatment enhanced high potassium-induced [³H]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25. A phosphorylation is likely to occur at Ser¹⁸⁷, as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins. SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co-immunoprecipitated with SNAP-25 was decreased by phosphorylation. These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.

Footnotes

↵* This work was supported by grant for Scientific Research on Priority Areas on “Functional Development of Neural Circuits,” the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PKC

protein kinase C

4α-phorbol

4α-phorbol-12,13-didecanoate

BoNT/A and BoNT/E

botulinum types A and B neurotoxin

mAb

monoclonal antibody

NE

norepinephrine

NSF

N-ethylmaleimide-sensitive fusion protein

PAGE

polyacrylamide gel electrophoresis

SNAPs

soluble NSF attachment proteins

SNAP-25

25-kDa synaptosome-associated protein

SNARE

SNAP receptor

PMA

phorbol 12-myristate 13-acetate

ATPγS

adenosine 5′-O-(thiotriphosphate).
- Received December 20, 1995.
- Revision received March 6, 1996.