STAT3 AND G-CSF-INDUCED MYELOID DIFFERENTIATION

Granulocyte colony-stimulating factor (G-CSF) is the cytokine critical for directing neutrophilic granulocyte differentiation. Early G-CSF s ignaling events in myeloid cells involves activation of STATs, protein s that serve the dual function of signal transduction and activation o f transcription, especially the activation of Stat3. A dominant-negati ve mutant construct of Stat3 inhibited G-CSF-mediated neutrophilic dif ferentiation indicating that Stat3 is a essential component for drivin g the G-CSF-mediated differentiation program in myeloid cells. Three i soforms of Stat3 have been identified, alpha (p92), beta (p83) and gam ma (p72) each derived from a single gene. Stat3 alpha is the predomina nt isoform expressed in most cells. Stat3 beta is derived from Stat3 a lpha by alternative RNA splicing. Stat3 gamma is derived from Stat3 al pha by limited proteolysis. Mapping of Stat3 alpha and Stat3 beta acti vation in M1 murine myeloid leukemia cells revealed that their optimal activation required G-CSFR constructs containing both Y704 and Y744. These amino acid residues has previously been demonstrated to be essen tial for C-CSF-induced differentiation in this cells. Phosphopeptide a ffinity and phosphopeptide inhibition studies indicate that Stat3 alph a and Stat3 beta are recruited to the G-CSF receptor complex through t heir interaction with the receptor at phosphotyrosines Y704 and Y744. Y744 is followed at the +3 position by Cys (C). This sequence YXXC, re presents a novel motif implicated in the recruitment and activation of Stat3 alpha, Stat3 beta and Stat3 gamma by the hG-CSFR. Structurally, Stat3 alpha, Stat3 beta and Stat3 gamma differ from each other in the ir C-terminal transactivation domain. In the beta isoform, the Stat3 a lpha transactivation domain is replaced by 7 amino acid residues which enable Stat3 beta to interact with c-Jun. In the gamma isoform, the S tat3 alpha transactivation domain is removed by limited proteolysis cr eating a dominant negative isoform. In immature human myeloid cells ca pable of differentiating into neutrophils in response to G-CSF, G-CSF did not activate Stat3 alpha, rather, it activated predominantly Stat3 beta These findings combined with recent reports linking Stat3a with proliferation and transformation suggest that the beta isoform of Stat 3 may be more critical for G-CSF-mediated differentiation. Activation of Stat3 gamma occurred predominantly in terminally differentiated neu trophils suggesting that it may be part of a controlled proteolytic me chanism modulating pro-proliferative protein(s) in mature myeloid cell s.