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 from the receptor for erythropoietin (Epo), although the Epo receptor (EpoR) lacks the tyrosine kinase domain. We have previously shown that the Jak2 tyrosine kinase couples with the EpoR to transduce a growth signal. In the present study, we demonstrate that Lyn, a Src family tyrosine kinase, physically associates with the EpoR in Epo-dependent hematopoietic cell lines, 32D/EpoR-Wt and F36E. Coexpression experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of 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 proteins further showed that the Src homology (SH)-2 domain of Lyn specifically 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 phosphopeptide competition assays further indicated that the Lyn SH2 domain directly binds to the tyrosine-phosphorylated EpoR, most likely through its interaction with phosphorylated Y-464 or Y-479 in the carboxy-terminal region of the EpoR. In vitro binding studies also demonstrated that 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 activates the DNA-binding and transcription-activating abilities of Stat5. In agreement with this, Lyn enhanced the Stat5-dependent transcriptional activation when overexpressed in 32D/EpoR-Wt cells. In addition, Lyn was demonstrated to phosphorylate the EpoR and Stat5 on tyrosines in vitro. These results suggest that Lyn may play a role in activation of the Jak2/Stat5 and other signaling pathways by the EpoR.
Chin
H
H
First Department of Internal Medicine and School of Allied Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
Arai
A
A
Wakao
H
H
Kamiyama
R
R
Miyasaka
N
N
Miura
O
O
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D005786:Gene Expression Regulation / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism; D007377:Interleukin-3 / Q000494:pharmacology; D053614:Janus Kinase 2; D051379:Mice; D008894:Milk Proteins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
D004268:DNA-Binding Proteins; D007377:Interleukin-3; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D019000:Phosphotyrosine; D004247:DNA; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases