IDENTIFICATION OF PHOSPHOPROTEINS SPECIFIC TO GRANULOCYTE-COLONY-STIMULATING FACTOR-MEDIATED SIGNALING USING 2D-SDS-PAGE

Like other cytokines, granulocyte colony-stimulating factor (G-CSF) ac tivates a complex array of signal transduction pathways involving mult iple kinases and phosphatases. We sought to identify phosphoproteins s pecific to G-CSF signaling. Using 2D-SDS-PAGE of P-32-labeled cytosoli c extracts, we compared phosphoprotein patterns of NFS-60 cells treate d with G-CSF or interleukin-3 (IL-3). We also compared the patterns fo und 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-rela ted 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 c ytokines was confirmed by in vitro kinase assays. The 2D-SDS-PAGE appr oach was also used to demonstrate that a series of unrelated G(1) phas e 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 demon strated by both in vitro kinase assays and 2D-SDS-PAGE. Therefore, 2D- SDS-PAGE has potential to dissect both the signal transduction pathway s lying downstream of the G-CSF receptor (and of the receptors for oth er CSFs) and also the site of action of proliferation inhibitors.