Granulocyte-macrophage colony-stimulating factor induces a staurosporine inhibitable tyrosine phosphorylation of unique neutrophil proteins.
Human neutrophils treated with chemotactic peptides or phorbol esters demonstrate tyrosine phosphorylation of a subset of proteins. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced a time- and concentration-dependent increase in the tyrosine phosphorylation of at least seven proteins. Three of these proteins with approximate molecular weights of 150, 95, and 70 Kd were unique to neutrophils treated with GM-CSF, and were not seen to be phosphorylated on tyrosine in neutrophils treated with the agonists FMLP or PMA, or the cytokines G-CSF and tumor necrosis factor. We found the 150-Kd protein to be localized within the cell particulate fraction and the 95-Kd protein within the cell cytosol. The 70-Kd phosphotyrosine protein was found in both fractions. When the neutrophils were treated with Triton X-100 (Sigma Chemical Co, St Louis, MO) to evaluate cytoskeletal associations of proteins, the 150 phosphotyrosine protein partitioned with the Triton X-100 insoluble cytoskeleton (TICS), and the 70-Kd protein partitioned with both the TICS and Triton X-100 soluble proteins. The GM-CSF-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor ST638. This was not seen with the putative C-kinase inhibitor, H-7. However, staurosporine was seen to inhibit tyrosine phosphorylation of neutrophil proteins by GM-CSF and in vitro tyrosine kinase activity of isolated neutrophil cytosol and particulate fractions. These data indicate that the three unique GM-CSF-induced phosphotyrosine-containing proteins may be responsible for the unique actions of GM-CSF and that staurosporine inhibits a tyrosine kinase responsible for the phosphorylation of these proteins.
Berkow
R L
RL
Department of Pediatrics, Pediatric Hematology/Oncology, University of Alabama, Birmingham 35233.
D000470:Alkaloids / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D000470:Alkaloids; D011506:Proteins; D014443:Tyrosine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate