Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphorylation in response to colony-stimulating factor 1/c-fms signaling.
An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation 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 response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein 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 kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA 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 kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.
Fowles
L F
LF
Departments of Microbiology and Biochemistry and the Centre for Molecular and Cellular Biology, University of Queensland, Queensland Q4072, Australia.
Martin
M L
ML
Nelsen
L
L
Stacey
K J
KJ
Redd
D
D
Clark
Y M
YM
Nagamine
Y
Y
McMahon
M
M
Hume
D A
DA
Ostrowski
M C
MC
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010766:Phosphorylation; D010769:Phosphothreonine / Q000032:analysis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012097:Repressor Proteins; D015534:Trans-Activators / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494069:ETS2 protein, human; C494070:Ets2 protein, mouse; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D010769:Phosphothreonine; D016173:Macrophage Colony-Stimulating Factor; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; D016186:Receptor, Macrophage Colony-Stimulating Factor; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases