PHYSICAL AND FUNCTIONAL INTERACTIONS BETWEEN STAT5 AND THE TYROSINE-PHOSPHORYLATED RECEPTORS FOR ERYTHROPOIETIN AND INTERLEUKIN-3

Erythropoietin (Epo) and interleukin-3 (IL-3) stimulate activation of the Jak2 tyrosine kinase and induce tyrosine phosphorylation and activ ation of Stat5, In the present study, we have shown that Epo or IL-3 s timulation induces binding of Stat5 to the tyrosine-phosphorylated Epo receptor (EpoR) or IL-3 receptor beta subunit (beta(IL3)), respective ly, in IL-3-dependent 32D cells expressing the EpoR. The binding of St at5 to these cytokine receptors was shown to be rapid and transient, o ccurring within 1 minute of stimulation of cells and significantly dec reasing after 5 minutes of cell treatment. In vivo binding experiments in COS cells showed that binding of Stat5 to the EpoR was mediated th rough the Stat5 Src homology 2 (SH2) domain. In vitro binding studies further showed that Stat5, but not other Stats examined, bound specifi cally to tyrosine-phosphorylated recombinant EpoR fusion proteins. In these in vivo and in vitro binding studies, Stat5 bound, albeit to a l esser degree, to truncated EpoR mutants in which all the intracellular tyrosines except Y-343 were removed. Furthermore, EpoR-derived synthe tic phosphotyrosine peptides corresponding to Y-343, Y-401, Y-431, and Y-479 inhibited the in vitro binding of Stat5. When expressed in 32D cells, a mutant EpoR in which all the intracellular tyrosines were rem oved by carboxy-terminal truncation showed a significantly impaired ab ility to induce tyrosine phosphorylation of Stat5, particularly at low concentrations of Epo, but exhibited an increased sensitivity to Epo for growth signaling as compared with the wild-type EpoR. These result s indicate that Stat5 specifically and transiently binds to the EpoR t hrough the interaction between the Stat5 SH2 domain and specific phosp horylated tyrosines, including Y-343, in the EpoR cytoplasmic domain. It was implied that beta(IL3) may also have similar Stat5 docking site s. The Stat5 docking sites in the EpoR were shown to facilitate specif ic activation of Stat5, which, however, may not be required for the Ep oR-mediated growth signaling. (C) 1996 by The American Society of Hema tology.