Identification of a novel Stat3 recruitment and activation motif within the granulocyte colony-stimulating factor receptor

Stat3 is essential for early embryonic development and for myeloid differen tiation induced by the cytokines granulocyte colony-stimulating factor (G-C SF) and interleukin-6 (IL-6). Two isoforms of Stat3 have been identified, a lpha (p92) and beta (p83), which have distinct transcriptional and biologic al functions. Activation of both Stat3 alpha and Stat3 beta requires the di stal cytoplasmic domain of the G-CSFR, which contains four Tyr at positions 704, 729, 744, and 764. The studies reported here were undertaken to deter mine which, if any, of these tyrosine residues participated in Stat3 alpha/ beta recruitment and activation. We showed that Stat3 alpha and Stat3 beta were affinity purified using phosphopeptides containing Y704 and Y744 but n ot by nonphosphorylated peptide analogues or by phosphopeptides containing Y729 and Y764. Complementary results were obtained in studies examining the ability of these peptides to destabilize and inhibit DNA binding of activa ted Stat3. Both Y704 and Y744 contributed to optimal activation of Stat3 al pha/beta in M1 murine myeloid leukemia cells containing wild-type and Y-to- F mutant G-CSFR constructs. Carboxy-terminal to Y704 at the +3 position is Gln; YXXQ represents a consensus Stat3 recruitment and activation motif. Y7 44 is followed at the +3 position by Cys (C): YXXC, represents a novel moti f implicated in the recruitment and activation of Stat3. Modeling of the SH 2 domain of Stat3 based on homologous SH2 domains of known structure reveal ed polar residues whose side chains contact the +3 position. This substitut ion may confer specificity for the Y704- and Y744-based ligands by allowing H-bond formation between the binding surface and the Gin or Cys found at t he respective +3 position. (C) 1999 by The American Society of Hematology.