PERSISTENT ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASES P42 AND P44 AND ETS-2 PHOSPHORYLATION IN RESPONSE TO COLONY-STIMULATING FACTOR 1C-FMS SIGNALING/

An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 i n response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Pho sphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate early respons e gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincide nt with expression of ets-2 protein. In fibroblasts, ets-2 was constit utively expressed and rapidly became phosphorylated in response to CSF -1. In both cell systems, ets-2 phosphorylation was persistent, with m aximal phosphorylation detected 8 to 2 1 h after CSF-1 stimulation, an d was correlated with activation of the CSF-1 target urokinase plasmin ogen activator (uPA) gene. Kinase assays that used recombinant ets-2 p rotein as a substrate demonstrated that mitogen-activated protein (MAP ) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinas es are the major ets-2 kinases activated in response to CSF-1/c-fms si gnaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as u PA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1 stimulated uPA reporter gene activity . These results indicate that persistent activation of the raf/MAP kin ase pathway by CSF-1 is necessary for both ets-2 expression and posttr anslational activation in macrophages.