Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

doi:10.1083/jcb.200308110

Published online 17 November 2003. doi:10.1083/jcb.200308110

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© The Rockefeller University Press, 0021-9525/2003/11/813 $8.00
The Journal of Cell Biology, Volume 163, Number 4, 813-824

Article

Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

Kazuhito Tomizawa¹, Satoshi Sunada¹, Yun-Fei Lu⁷, Yoshiya Oda⁴, Masahiro Kinuta², Toshio Ohshima⁵, Taro Saito⁶, Fan-Yan Wei¹, Masayuki Matsushita¹, Sheng-Tian Li¹, Kimiko Tsutsui³, Shin-ichi Hisanaga⁶, Katsuhiko Mikoshiba⁵, Kohji Takei² and Hideki Matsui¹^,7

¹ Department of Physiology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
² Department of Neuroscience, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
³ Department of Neuroanatomy, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
⁴ Laboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki 300-2635, Japan
⁵ Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Saitama 351-0198, Japan
⁶ Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan
⁷ Protein Therapy, New Techno-Venture Oriented R&D, Japan Science and Technology Corporation, Okayama 700-8558, Japan

Address correspondence to Kazuhito Tomizawa, Department of Physiology, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho 2-5-1, Okayama 700-8558, Japan. Tel.: 81-86-235-7109. Fax: 81-86-235-7111. email: tomikt{at}md.okayama-u.ac.jp

It has been thought that clathrin-mediated endocytosis is regulatedby phosphorylation and dephosphorylation of many endocytic proteins,including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependentcophosphorylation of amphiphysin I and dynamin I plays a criticalrole in such processes. Cdk5 inhibitors enhanced the electricstimulation–induced endocytosis in hippocampal neurons,and the endocytosis was also enhanced in the neurons of p35-deficientmice. Cdk5 phosphorylated the proline-rich domain of both amphiphysinI and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylationof amphiphysin I inhibited the association with ß-adaptin.Furthermore, the phosphorylation of dynamin I blocked its bindingto amphiphysin I. The phosphorylation of each protein reducedthe copolymerization into a ring formation in a cell-free system.Moreover, the phosphorylation of both proteins completely disruptedthe copolymerization into a ring formation. Finally, phosphorylationof both proteins was undetectable in p35-deficient mice.

Key Words: endocytic protein; p35; cyclin-dependent kinase; presynapse; synaptosome

The online version of this article includes supplemental material.

Abbreviations used in this paper: DLS, dynamic light scattering;ESI, electrospray ionization; LC, liquid chromatography; MALDI-MS,matrix assisted laser desorption/ionization mass spectrometry;PRD, proline-rich domain; SH3, Src homology 3; VDCC, voltage-dependentcalcium channel.

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