Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphorylation in response to colony-stimulating factor 1/c-fms signaling

L F Fowles, M L Martin, L Nelsen, K J Stacey, D Redd, Y M Clark, Y Nagamine, M McMahon, D A Hume, M C Ostrowski

Research output: Contribution to journalArticlepeer-review

Abstract

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.
Original languageEnglish
Pages (from-to)5148-56
Number of pages9
JournalMolecular and Cellular Biology
Volume18
Issue number9
Publication statusPublished - Sep 1998

Keywords

  • 3T3 Cells
  • Animals
  • Blotting, Western
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Cell Line
  • DNA-Binding Proteins
  • Enzyme Activation
  • Humans
  • Kinetics
  • Luciferases
  • Macrophage Colony-Stimulating Factor
  • Macrophages
  • Mice
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases
  • Phosphorylation
  • Phosphothreonine
  • Protein-Tyrosine Kinases
  • Proto-Oncogene Protein c-ets-2
  • Proto-Oncogene Proteins
  • RNA, Messenger
  • Receptor, Macrophage Colony-Stimulating Factor
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Trans-Activators
  • Transcription Factors
  • Transcription, Genetic
  • Transfection

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