Identification of tyrosine-phosphorylated colony-stimulating factor 1 (CSF-1) receptor and a 56-kilodalton protein phosphorylated in intact human cells in response to CSF-1.
Colony-stimulating factor 1 (CSF-1) selectively supports the survival, proliferation, and maturation of hemopoietic cells of the monocyte/macrophage lineage. Although the cellular receptor for CSF-1, (the c-fms protein) is a protein-tyrosine kinase activated by the binding of CFS-1, the role of phosphorylation of cellular proteins in CSF-1 signal transduction is poorly understood. Therefore, we examined the CSF-1-stimulated phosphorylation of cellular proteins in human BeWo choriocarcinoma cell line (known to express the c-fms protein). BeWo cells were metabolically labeled with 32Pi, stimulated with recombinant human CSF-1, and extracted with detergent. Phosphotyrosyl proteins were isolated from detergent extracts by affinity chromatography on a highly specific antibody to phosphotyrosine. Rapid phosphorylation of 170-kd protein, followed closely by the phosphorylation of a 56-kd protein, was observed in response to CSF-1. The 170-kd phosphotyrosyl protein bound to wheat germ agglutinin and was secondarily immunoprecipitated with a specific anti-fms serum, consistent with its identity as the CSF-1 receptor. Although purified human macrophages that proliferate in culture in response to CSF-1 are not generally accessible, CSF-1 did stimulate the phosphorylation of a 56-kd protein in intact mononuclear leukocytes from human peripheral blood. Thus, the BeWo cell line may represent a good model for the study of CSF-1-stimulated cellular protein phosphorylation.
Huhn
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Department of Medicine, Roger Williams General Hospital, Brown University, Providence, Rhode Island 02908.
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Frackelton
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D002478:Cells, Cultured; D003115:Colony-Stimulating Factors / Q000032:analysis* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D016173:Macrophage Colony-Stimulating Factor; D010761:Phosphorus Radioisotopes; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D011506:Proteins / Q000302:isolation & purification*; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification*; D016186:Receptor, Macrophage Colony-Stimulating Factor; D015398:Signal Transduction / Q000187:drug effects; D012967:Sodium Dodecyl Sulfate; D014443:Tyrosine / Q000031:analogs & derivatives
D003115:Colony-Stimulating Factors; D010761:Phosphorus Radioisotopes; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D012967:Sodium Dodecyl Sulfate; D014443:Tyrosine; D016173:Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; D016186:Receptor, Macrophage Colony-Stimulating Factor