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J. Biol. Chem., Vol. 275, Issue 36, 27775-27783, September 8, 2000
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From the Nonstructural protein 3 (Nsp3) is an essential
subunit of the alphavirus RNA replication complex, although its
specific function(s) has yet to be well defined. Previously, it has
been shown that Semliki Forest virus Nsp3 (482 amino acids) is a
phosphoprotein, and, in the present study, we have mapped its major
phosphorylation sites. Mass spectrometric methods utilized included
precursor ion scanning, matrix-assisted laser desorption/ionization
mass spectrometry used in conjunction with on-target alkaline
phosphatase digestions, and tandem mass spectrometry. Two-dimensional
peptide mapping was applied to separate tryptic
32P-labeled phosphopeptides of Nsp3. Radiolabeled
peptides were then subjected to Edman sequencing, and phosphoamino acid
analysis. In addition, radiolabeled Nsp3 was cleaved successively with
cyanogen bromide and trypsin, and microscale iron-chelate affinity
chromatography was used to enrich phosphopeptides. By combining these
methods, we showed that Nsp3 is phosphorylated on serine residues 320, 327, 332, 335, 356, 359, 362, and 367, and is heavily phosphorylated on
peptide Gly338-Lys415, which carries 7-12
phosphates distributed over its 13 potential phosphorylation sites.
These analytical findings were corroborated by constructing a Nsp3
derivative devoid of phosphorylation. The results represent the first
determination of phosphorylation sites of an alphavirus nonstructural
protein, but the approach can be utilized in phosphoprotein analysis in general.
Phosphorylation Site Analysis of Semliki Forest Virus
Nonstructural Protein 3*
§ and Program in Cellular Biotechnology and
¶ Protein Chemistry Laboratory, Institute of Biotechnology,
Viikki Biocenter, University of Helsinki,
Helsinki FIN-00014, Finland
*
This work was supported by Academy of Finland Grant
8397, the Technology Development Center, and the Helsinki
University Foundation.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.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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