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Molecular & Cellular Proteomics 7:2123-2137, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Proteomics Analysis Identifies Phosphorylation-dependent -Synuclein Protein Interactions ^*,S

Melinda A. McFarland^,, Christopher E. Ellis^,¶, Sanford P. Markey and Robert L. Nussbaum^||,**

From the National Institute of Mental Health and ^¶ NHGRI, National Institutes of Health, Bethesda, Maryland 20891 and the ^|| Department of Medicine, Division of Medical Genetics, University of California, San Francisco, California 94143

Mutations and copy number variation in the SNCA gene encoding the neuronal protein -synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183–R194). The carboxyl terminus of -synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated -synuclein. J. Biol. Chem. 275, 390–397). Under pathological conditions, such as in Parkinson disease, -synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of -synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739–29752). Controversy exists over the extent to which phosphorylation of -synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated -synuclein. We showed that non-phosphorylated -synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas -synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated -synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when -synuclein is phosphorylated.

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