LYN PHYSICALLY ASSOCIATES WITH THE ERYTHROPOIETIN RECEPTOR AND MAY PLAY A ROLE IN ACTIVATION OF THE STAT5 PATHWAY

Protein tyrosine phosphorylation plays a crucial role in signaling fro m the receptor for erythropoietin (Epo), although the Epo receptor (Ep oR) lacks the tyrosine kinase domain. We have previously shown that th e Jak2 tyrosine kinase couples with the EpoR to transduce a growth sig nal. In the present study, we demonstrate that Lyn, a Src family tyros ine kinase, physically associates with the EpoR in Epo-dependent hemat opoietic cell lines, 32D/EpoR-Wt and F36E. Coexpression experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation o f the EpoR and that both LynA and LynB, alternatively spliced forms of Lyn, bind with the membrane-proximal 91-amino acid region of the EpoR cytoplasmic domain. In vitro binding studies using GST-Lyn fusion pro teins further showed that the Src homology (SH)-2 domain of Lyn specif ically binds with the tyrosine-phosphorylated EpoR in lysate from Epo- stimulated cells, whereas the tyrosine kinase domain of Lyn binds with the unphosphorylated EpoR. Far-Western blotting and synthetic phospho peptide competition assays further indicated that the Lyn SH2 domain d irectly binds to the tyrosine-phosphorylated EpoR, most likely through its interaction with phosphorylated Y-464 or Y-479 in the carboxy-ter minal region of the EpoR. In vitro binding studies also demonstrated t hat the Lyn SH2 domain directly binds to tyrosine-phosphorylated Jak2. In vitro reconstitution experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of Stat5, mainly on Y-694, and a ctivates the DNA-binding and transcription-activating abilities of Sta t5. In agreement with this, Lyn enhanced the State-dependent transcrip tional activation when overexpressed in 32D/EpoR-Wt cells. In addition , Lyn was demonstrated to phosphorylate the EpoR and Stat5 on tyrosine s in vitro. These results suggest that Lyn may play a role in activati on of the Jak2/Stat5 and other signaling pathways by the EpoR. (C) 199 8 by The American Society of Hematology.