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

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Abstract
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.