Identification of phosphoproteins specific to granulocyte colony-stimulating factor-mediated signaling using 2D-SDS-PAGE.
Like other cytokines, granulocyte colony-stimulating factor (G-CSF) activates a complex array of signal transduction pathways involving multiple kinases and phosphatases. We sought to identify phosphoproteins specific to G-CSF signaling. Using 2D-SDS-PAGE of 32P-labeled cytosolic extracts, we compared phosphoprotein patterns of NFS-60 cells treated with G-CSF or interleukin-3 (IL-3). We also compared the patterns found after stimulation of M-NFS-60 cells with macrophage-CSF (M-CSF). A large number of phosphoproteins were found that were specific for the G-CSF response. Their distribution contrasted with that of Erk-1-related spots, identified by Western blotting, which were common to G-CSF, M-CSF (CSF-1), and IL-3 responses. The activation of Erk-1 by these cytokines was confirmed by in vitro kinase assays. The 2D-SDS-PAGE approach was also used to demonstrate that a series of unrelated G1 phase inhibitors of the mitogenic action of G-CSF elicited both common and diverse protein phosphorylation changes in G-CSF-treated NFS-60 cells that were not dependent on the inhibition of Erk-1 activity, as demonstrated by both in vitro kinase assays and 2D-SDS-PAGE. Therefore, 2D-SDS-PAGE has potential to dissect both the signal transduction pathways lying downstream of the G-CSF receptor (and of the receptors for other CSFs) and also the site of action of proliferation inhibitors.
Csar
X F
XF
University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Victoria, Australia.
Ward
A C
AC
Hoffmann
B W
BW
Guy
G G
GG
Hamilton
J A
JA
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002457:Cell Extracts; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D015180:Electrophoresis, Gel, Two-Dimensional; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006131:Growth Inhibitors / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D011994:Recombinant Proteins; D015398:Signal Transduction / Q000187:drug effects*
D002457:Cell Extracts; D006131:Growth Inhibitors; D007377:Interleukin-3; D010750:Phosphoproteins; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D016173:Macrophage Colony-Stimulating Factor; D004247:DNA; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases