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.