Phosphorylation site analysis of Semliki forest virus nonstructural protein 3

Anja Helena Vihinen, Juhani Saarinen

Forskningsoutput: TidskriftsbidragArtikelVetenskapligPeer review

Sammanfattning

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 (32)P-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 Gly(338)-Lys(415), 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.
Originalspråkengelska
TidskriftJournal of Biological Chemistry
Volym36
Sidor (från-till)27775-83
Antal sidor9
ISSN0021-9258
StatusPublicerad - 8 sep 2000
MoE-publikationstypA1 Tidskriftsartikel-refererad

Citera det här

@article{860aa2c9b1c74f1c98cc4d2ce091fed0,
title = "Phosphorylation site analysis of Semliki forest virus nonstructural protein 3",
abstract = "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 (32)P-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 Gly(338)-Lys(415), 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.",
author = "Vihinen, {Anja Helena} and Juhani Saarinen",
year = "2000",
month = "9",
day = "8",
language = "English",
volume = "36",
pages = "27775--83",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology",

}

Phosphorylation site analysis of Semliki forest virus nonstructural protein 3. / Vihinen, Anja Helena; Saarinen, Juhani.

I: Journal of Biological Chemistry, Vol. 36, 08.09.2000, s. 27775-83.

Forskningsoutput: TidskriftsbidragArtikelVetenskapligPeer review

TY - JOUR

T1 - Phosphorylation site analysis of Semliki forest virus nonstructural protein 3

AU - Vihinen, Anja Helena

AU - Saarinen, Juhani

PY - 2000/9/8

Y1 - 2000/9/8

N2 - 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 (32)P-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 Gly(338)-Lys(415), 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.

AB - 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 (32)P-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 Gly(338)-Lys(415), 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.

M3 - Article

VL - 36

SP - 27775

EP - 27783

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

ER -