GRANULOCYTE-COLONY-STIMULATING FACTOR RAPIDLY ACTIVATES A DISTINCT STAT-LIKE PROTEIN IN NORMAL MYELOID CELLS

Binding of granulocyte colony-stimulating factor (G-CSF) to normal mye loid cells activates the protein tyrosine kinases Lyn and Syk and resu lts in the immediate early upregulation of G-CSF receptor (R) mRNA. In our studies of the signaling pathways activated by G-CSF that are cou pled to proliferation and differentiation of myeloid cells, we examine d whether G-CSF activated a latent transcription factor belonging to t he STAT protein family. Electrophoretic mobility shift assays (EMSAs) of nuclear extracts from G-CSF-stimulated human myeloid cells showed t he rapid activation of a DNA-binding protein that bound to the high-af finity serum-inducible element (hSIE) and migrated with mobility simil ar to serum inducible factor (SIF)-A (Stat3 homodimer). The G-CSF-stim ulated SIF-A complex (G-SIF-A) did not bind to duplex oligonucleotides used to purify and characterize other Stat proteins (Stat1-6). In add ition, antibodies raised against Stat1-6 failed to supershift the G-SI F-A complex or interfere with its formation. Based on its binding to t he hSIE and lack of antigenic cross-reactivity with other known STAT p roteins that bind to this element, it is likely that G-SIF-A is compos ed of a distinct member of the STAT protein family. EMSAs of whole-cel l extracts prepared from cell lines containing full-length and truncat ed mutants of the G-CSFR showed that activation of G-SIF-A did not cor relate with proliferation; rather, optimal activation requires the dis tal half of the cytosolic domain of the G-CSFR that is essential for d ifferentiation. Activation of G-SIF-A, therefore, may be an early G-CS FR-coupled event that is critical for myeloid maturation. (C) 1995 by The American Society of Hematology.