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.
Csar
X F
XF
University of Melbourne, Arthritis and Inflammation Research Centre, Department of Medicine, Royal Melbourne Hospital, Parkville 3050, Victoria, Australia. xfc@unimelb.edu.au
Wilson
N J
NJ
Strike
P
P
Sparrow
L
L
McMahon
K A
KA
Ward
A C
AC
Hamilton
J A
JA
D000818:Animals; D002460:Cell Line; D002845:Chromatography; D017886:Durapatite; D015180:Electrophoresis, Gel, Two-Dimensional; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D007377:Interleukin-3 / Q000494:pharmacology; D007526:Isoelectric Point; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification; D010766:Phosphorylation; D020543:Proteome; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000187:drug effects* / Q000302:isolation & purification
D007377:Interleukin-3; D010750:Phosphoproteins; D020543:Proteome; D016179:Granulocyte Colony-Stimulating Factor; D017886:Durapatite; D013482:Superoxide Dismutase