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Identification of phosphoproteins specific to granulocyte colony-stimulating factor-mediated signaling using 2D-SDS-PAGE
journal contributionposted on 1997-01-01, 00:00 authored by X F Csar, Alister WardAlister Ward, B W Hoffmann, G G Guy, J A Hamilton
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.
JournalJournal of interferon and cytokine research
Pagination77 - 86
PublisherMary Ann Liebert Publishers
LocationNew Rochelle, N.Y.
Publication classificationC1.1 Refereed article in a scholarly journal
Copyright notice1997, Mary Ann Liebert Publishers
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AnimalsCalcium-Calmodulin-Dependent Protein KinasesCell DivisionCell ExtractsCell LineCytosolDNAElectrophoresis, Gel, Two-DimensionalGranulocyte Colony-Stimulating FactorGrowth InhibitorsInterleukin-3Macrophage Colony-Stimulating FactorMiceMitogen-Activated Protein Kinase 3Mitogen-Activated Protein KinasesPhosphoproteinsPhosphorylationRecombinant ProteinsSignal TransductionScience & TechnologyLife Sciences & BiomedicineBiochemistry & Molecular BiologyCell BiologyImmunologyTUMOR-NECROSIS-FACTORFACTOR-RECEPTORTYROSINE PHOSPHORYLATIONHUMAN NEUTROPHILSACTIVATIONPROTEINCELLSDIFFERENTIATIONPROLIFERATIONINHIBITION