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Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells.
journal contributionposted on 2001-01-01, 00:00 authored by X Csar, N Wilson, P Strike, L Sparrow, K McMahon, Alister WardAlister Ward, J Hamilton
Using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) of 32P-labeled cytosolic and membrane extracts, we identified a 21.5 kDa phosphoprotein with an isoelectric point of 6.0 in NFS-60 cells that was phosphorylated maximally at 15 min by treatment with granulocyte-colony stimulating factor (G-CSF) but not with interlevkin-3 (IL-3) or colony-stimulating factor-1 (macrophage-colony stimulating factor (CSF-1 (M-CSF)). The phosphorylation of this protein, designated 21.5/6.0, was unaffected by a series of antiproliferative agents . These findings suggested that the 21.5/6.0 phosphoprotein may be involved in specific G-CSF-mediated biological responses such as activation and/or differentiation. We sought to characterize this 21.5/6.0 by a novel combination of 2-D SDS-PAGE and hydroxyapatite (HTP)-chromatography. Amino acid sequence determination of 21.5/6.0 revealed it to share a high level of homology with copper/zinc superoxide dismutase (Cu/Zn-SOD), indicating that a Cu/Zn-SOD is phosphorylated following treatment with G-CSF. This is the first report of the phosphorylation and possible involvement of Cu/Zn-SOD protein in granulocyte activation/differentiation events. In addition, Cu/Zn-SOD levels and activity were diminished by G-CSF but not IL-3 treatment. This new protocol combining 2-D SDS-PAGE and HTP-chromatography allows the characterization of low abundance phosphoproteins involved in the cellular responses to G-CSF and presumably to other cytokines/growth factors.
Pagination435 - 443
PublisherWiley - V C H Verlag
Publication classificationC1.1 Refereed article in a scholarly journal
Copyright notice2001, Wiley
Read the peer-reviewed publication
Superoxide dismutaseMicrosequencingHydroxyapatiteProtein purificationProtein phosphorylationTwo-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresisMacrophage-colony stimulating factorGranulocyte-colony stimulating factor 1Science & TechnologyLife Sciences & BiomedicineBiochemical Research MethodsBiochemistry & Molecular Biologygranulocyte-colony stimulating factor granulocyte-colony stimulating factor 1 macrophage-colony stimulating factorTUMOR-NECROSIS-FACTORFACTOR-RECEPTORTYROSINE PHOSPHORYLATIONHEMATOPOIETIC-CELLSSIGNAL-TRANSDUCTIONGENE-EXPRESSIONG-CSFPROTEINSACTIVATIONDIFFERENTIATION