title: Tyrosine-dependent and -independent mechanisms of STAT3 activation by the human granulocyte colony-stimulating factor (G-CSF) receptor are differentially utilized depending on G-CSF concentration.
abstract: The granulocyte colony-stimulating factor receptor (G-CSF-R) activates multiple STAT proteins. Although the membrane-proximal cytoplasmic region of the G-CSF-R is necessary and sufficient for activation of STAT1 and STAT5, activation of STAT3 requires the membrane distal region that contains four tyrosines. Although one of these (Y704) has previously been shown to be involved in STAT3 activation from a truncated G-CSF-R derived from a patient with severe chronic neutropenia (SCN), this tyrosine is not required for STAT3 activation by the full-length G-CSF-R. To investigate possible alternative mechanisms of STAT3 activation, we generated a series of Ba/F3 cell transfectants expressing the wild-type G-CSF-R or mutant receptors that either completely lack tyrosines or retain just one of the four cytoplasmic tyrosines of the G-CSF-R. We show that, at saturating G-CSF concentrations, STAT3 activation from the full-length G-CSF-R is efficiently mediated by the C-terminal domain in a manner independent of receptor tyrosines. In contrast, at low G-CSF concentrations, Y704 and Y744 of the G-CSF-R play a major role in STAT3 activation. Both tyrosine-dependent and -independent mechanisms of STAT3 activation are sensitive to the Jak2 inhibitor AG-490, follow similar kinetics, and lead to transactivation of a STAT3 reporter construct, indicating functional equivalence. STAT3 activation is also impaired, particularly at nonsaturating G-CSF concentrations, in bone marrow cells from mice expressing a truncated G-CSF-R (gcsfr-triangle up715). These findings suggest that G-CSF-induced STAT3 activation during basal granulopoiesis (low G-CSF) and "emergency" granulopoiesis (high G-CSF) are differentially controlled. In addition, the data establish the importance of the G-CSF-R C-terminus in STAT3 activation in primary cells, which has implications for understanding why truncated G-CSF-R derived from SCN patients are defective in maturation signaling.
authors.affiliation: Institute of Hematology, Erasmus University, Rotterdam, The Netherlands. ward@hema.fgg.eur.nl
authors.forename: A C
authors.initials: AC
authors.lastname: Ward
authors.forename: M H
authors.initials: MH
authors.lastname: Hermans
authors.forename: L
authors.initials: L
authors.lastname: Smith
authors.forename: Y M
authors.initials: YM
authors.lastname: van Aesch
authors.forename: A M
authors.initials: AM
authors.lastname: Schelen
authors.forename: C
authors.initials: C
authors.lastname: Antonissen
authors.forename: I P
authors.initials: IP
authors.lastname: Touw
mesh_terms: D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D009503:Neutropenia / Q000235:genetics / Q000378:metabolism; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D016179:Granulocyte Colony-Stimulating Factor; D014443:Tyrosine