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J. Biol. Chem., Vol. 282, Issue 20, 14695-14707, May 18, 2007

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The in Vivo Phosphorylation Sites of Rat Brain Dynamin I^*

Mark E. Graham, Victor Anggono, Nicolai Bache, Martin R. Larsen, George E. Craft, and Phillip J. Robinson¹

From the Cell Signaling Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia and the Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark

Dynamin I (dynI) is phosphorylated in synaptosomes at Ser⁷⁷⁴ and Ser⁷⁷⁸ by cyclin-dependent kinase 5 to regulate recruitment of syndapin I for synaptic vesicle endocytosis, and in PC12 cells on Ser⁸⁵⁷. Hierarchical phosphorylation of Ser⁷⁷⁴ precedes phosphorylation of Ser⁷⁷⁸. In contrast, Thr⁷⁸⁰ phosphorylation by cdk5 has been reported as the sole site (Tomizawa, K., Sunada, S., Lu, Y. F., Oda, Y., Kinuta, M., Ohshima, T., Saito, T., Wei, F. Y., Matsushita, M., Li, S. T., Tsutsui, K., Hisanaga, S. I., Mikoshiba, K., Takei, K., and Matsui, H. (2003) J. Cell Biol. 163, 813–824). To resolve the discrepancy and to better understand the biological roles of dynI phosphorylation, we undertook a systematic identification of all phosphorylation sites in rat brain nerve terminal dynI. Using phosphoamino acid analysis, exclusively phospho-serine residues were found. Thr⁷⁸⁰ phosphorylation was not detectable. Mutation of Ser⁷⁷⁴, Ser⁷⁷⁸, and Thr⁷⁸⁰ confirmed that Thr⁷⁸⁰ phosphorylation is restricted to in vitro conditions. Mass spectrometry of ³²P-labeled phosphopeptides separated by two-dimensional mapping revealed seven in vivo phosphorylation sites: Ser⁷⁷⁴, Ser⁷⁷⁸, Ser⁸²², Ser⁸⁵¹, Ser⁸⁵⁷, Ser⁵¹², and Ser³⁴⁷. Quantification of ³²P radiation in each phosphopeptide showed that Ser⁷⁷⁴ and Ser⁷⁷⁸ were the major sites (up to 69% of the total), followed by Ser⁸⁵¹ and Ser⁸⁵⁷ (12%), and Ser⁸⁵³ (2%). Phosphorylation of Ser⁸⁵¹ and Ser⁸⁵⁷ was restricted to the long tail splice variant dynIxa and was not hierarchical. Co-purified, ³²P-labeled dynIII was phosphorylated at Ser⁷⁵⁹, Ser⁷⁶³, and Ser⁸⁵³. Ser⁸⁵³ is homologous to Ser⁸⁵¹ in dynIxa. The results identify all major and several minor phosphorylation sites in dynI and provide the first measure of their relative abundance and relative responses to depolarization. The multiple phospho-sites suggest subtle regulation of synaptic vesicle endocytosis by new protein kinases and new protein-protein interactions. The homologous dynI and dynIII phosphorylation indicates a high mechanistic similarity. The results suggest a unique role for the long splice variants of dynI and dynIII in nerve terminals.

Received for publication, October 16, 2006 , and in revised form, February 12, 2007.

^* This work was supported by grants form the National Health and Medical Research Council of Australia (to P. J. R.) and University of Sydney Postgraduate Awards (to V. A. and G. E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed. Tel.: 61-2-9687-2800; Fax: 61-2-9687-2120; E-mail: probinson{at}cmri.com.au.

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