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 electrophor esis (2-D SDS-PAGE) of 32P-labeled cytosolic and membrane extracts, we iden tified 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 granul ocyte-colony stimulating factor (G-CSF) but not with interlevkin-3 (IL-3) o r colony-stimulating factor-1 (macrophage-colony stimulating factor(CSF-I ( M-CSF)). The phosphorylation of this protein, designated 21.5/6.0, was unaf fected by a series of antiproliferative agents [32]. These findings suggest ed that the 21.5/6.0 phosphoprotein may be involved in specific G-CSF-media ted biological responses such as activation and/or differentiation. We soug ht 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 sup eroxide dismutase (Cu/Zn-SOD), indicating that a Cu/Zn-SOD is phosphorylate d following treatment with G-CSF. This is the first report of the phosphory lation and possible involvement of CuZn-SOD protein in granulocyte activati on/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 abundan ce phosphoproteins involved in the cellular responses to G-CSF and presumab ly to other cytokines/growth factors.