Site-specific Phosphorylation of Synapsin I by Mitogen-activated Protein Kinase and Cdk5 and Its Effects on Physiological Functions

doi:10.1074/jbc.271.35.21108

Site-specific Phosphorylation of Synapsin I by Mitogen-activated Protein Kinase and Cdk5 and Its Effects on Physiological Functions*

From the ^‡ Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-11, Japan and the
^∥ Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo 194, Japan

^” To whom all correspondence should be addressed. Tel.: 81-562-93-9381; Fax: 81-562-93-8832.

↵^¶ Present address: Nippi Research Institute of Biomatrix, Adachi-ku, Tokyo 120, Japan.

Abstract

Posttranslational modifications of synapsin I, a major phosphoprotein in synaptic terminals, were studied by mass spectrometry. In addition to a well known phosphorylation site by calmodulin-dependent protein kinase II (CaM kinase II), a hitherto unrecognized site (Ser⁵⁵³) was found phosphorylated in vivo. The phosphorylation site is immediately followed by a proline, suggesting that the protein is an in vivo substrate of so-called proline-directed protein kinase(s). To identify the kinase involved, three proline-directed protein kinases expressed highly in the brain, i.e. mitogen-activated protein (MAP) kinase, Cdk5-p23, and glycogen synthase kinase 3β, were tested for the in vitro phosphorylation of synapsin I. Only MAP kinase and Cdk5-p23 phosphorylated synapsin I stoichiometrically. The phosphorylation sites were determined to be Ser⁵⁵¹ and Ser⁵⁵³ with Cdk5-p23, and Ser⁶², Ser⁶⁷, and Ser⁵⁵¹ with MAP kinase. Upon phosphorylation with MAP kinase, synapsin I showed reduced F-actin bundling activity, while no significant effect on the interaction was observed with the protein phosphorylated with Cdk5-p23. These results raise the possibility that the so-called proline-directed protein kinases together with CaM kinase II and cAMP-dependent protein kinase play an important role in the regulation of synapsin I function.

Footnotes

↵^§ A Postdoctoral Fellow of the Japan Society for the Promotion of Science.
↵* This work was supported in part by Grants-in-Aid from the Fujita Health University, Grants-in-Aid for Scientific Research (C) (06680773), and Grants-in-Aid for Scientific Research on Priority Areas (06253218, 06276218, 07268221, and 07279242) from the Ministry of Education, Science and Culture, 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:

CaM kinase

calmodulin-dependent protein kinase

LC/MS

liquid chromatography/mass spectrometry

MARCKS

myristoylated alanine-rich protein kinase C substrate, a major in vivo protein kinase C substrate protein

MAP kinase

mitogen-activated protein kinase

Cdk5-p23

cyclin-dependent protein kinase 5-p23 complex

GSK3β

glycogen synthase kinase 3β

PTH

phenylthiohydantoin

DTT-Ser

dithiothreitol adduct of dehydroalanine

Mes

4-morpholineethanesulfonic acid.
- Received January 29, 1996.
- Revision received June 10, 1996.