Structural Determinants for Signal Transducer and Activator of Transcription (STAT) 3 Recruitment and Activation by the Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) at Phosphotyrosine Ligands 704 and 744.

Four tyrosine (Y) residues within the cytoplasmic domain of the G-CSFR (Y704, Y228, Y744 and Y764 in the human receptor; Y703, Y227, Y743 and Y763 in the murine receptor) become phosphorylated by Jak kinases upon ligand binding leading to recruitment of Src homology (SH) 2 domain-containing proteins that link to programs for myeloid cell survival and differentiation (Stat3 recruitment to Y704 and Y744) and proliferation (SHP-2 and PI3K recruitment to Y04; Grb2, Shc and SHP-2 recruitment to Y764). While the preference of SH2 domain binding to specific phospho (p) Y peptide ligands was shown to map to the three residues immediately C-terminal to the pY (+1, +2, +3 residues), the structural basis for these preferences is poorly understood but could be exploited to specifically target deleterious G-CSFR-mediated signaling events such as aberrant Stat3 activation, which has been demonstrated in a subset of acute myelogenous leukemia (AML) patients whose cells contain Flt3 internal tandem duplications and who suffer relapse following initial chemotherapy. To establish the structural basis for Stat3 recruitment and activation by the G-CSFR at Y704 and Y744, we generated purified recombinant full-length Stat3 and phosphododecapeptides based on the sequence surrounding each Y within the G-CSFR. In peptide pull-down assays, recombinant Stat3 bound only to Y704 and Y744 phosphododecapeptide, which contain core pY motifs consisting of pYVLQ and pYLRC, respectively. In mirror resonance affinity assays employed to obtain quantitative binding information, Stat3 bound to each phosphododecapeptide with similar kinetics (e.g. K D s = 0.703 and 0.95 μM, respectively). We tested three models for Stat3 SH2-pY ligand binding proposed by us and others using wild type and mutant recombinant Stat3 proteins in peptide pull-down and mirror resonance affinity assays along with computer modeling of this interaction using the known structures of Statβ SH2 and EGFR pY ligand (EpY 1068 INQ). Our results revealed loss of binding of Stat3 to Y704 and Y744 phosphododecapeptides only in Stat3 mutated within the SH2 domain at K591 or R609, whose side chains interacted with the pY phosphate group, and in Stat3 mutated within the SH2 domain at E638, whose amide hydrogen bonded with oxygen within the +3 Q side chain (or with sulfur within the +3 C side chain) when the pY ligand assumes a β turn. G-CSF stimulation of cells co-expressing full-length G-CSFR and either wild type or mutant Stat3 constructs confirmed the requirements for the side chain of R609 and the amide hydrogen of E638 within the Stat3 SH2 domain for binding to the G-CSFR and subsequent phosphorylation of Stat3 on Y705. Thus, our findings identify for the first time the structural basis for recruitment and activation of Stat3 by the G-CSFR and reveal unique features of their interaction at Y704 and Y744 i.e. a β turn within the receptor pY motif and a key hydrogen bond formed between the polar side chain of the +3 residue and the amide hydrogen of E638 within the Stat3 SH2 domain. These features explain the preference of the Stat3 SH2 domain for pY peptide ligands with +3 Q or C as well as +3 T (pY 705 LKT within Stat3) and +3 S (pY 743 IRS within the murine G-CSFR) and can be exploited using a structure-assisted drug design strategy to develop new therapies for a subset of AML patients with poor prognosis whose cells demonstrate aberrant activation of Stat3.