Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells

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 [32]. 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.