Erythropoietin induces tyrosine phosphorylation of the interleukin-3 receptor beta subunit (betaIL3) and recruitment of Stat5 to possible Stat5-docking sites in betaIL3.
The receptors for erythropoietin (Epo) and interleukin-3 (IL-3) both induce the ligand-dependent activation of the Jak2 tyrosine kinase. Activated Jak2 then phosphorylates these receptors and thereby recruits various signaling molecules containing the Src homology (SH)-2 domain, including Stat5, to the tyrosine phosphorylated receptors. In the present study, we demonstrate that Epo stimulation induces unidirectional cross-phosphorylation of the IL-3 receptor beta subunit (betaIL3) on tyrosines and its rapid and transient association with Stat5 in murine IL-3-dependent cell lines engineered to express the Epo receptor (EpoR). Using cell lines expressing various EpoR mutants, it was demonstrated that the Epo-induced tyrosine phosphorylation of betaIL3 is dependent on the membrane-proximal EpoR cytoplasmic region involved in the activation of Jak2, but not on the extracellular and transmembrane regions or on the carboxy-terminal 145 amino acid region containing all the intracellular tyrosine residues. It was also shown that IL-3 induces rapid and dose-dependent association of Jak2 with betaIL3. However, Epo failed to induce any detectable association of betaIL3 with Jak2 or the EpoR. The present study also demonstrates that in IL-3-stimulated cells, an ovine Stat5 mutant harboring a substitution of Tyr694 to Phe, which abolishes the tyrosine phosphorylation required for activation, fails to dimerize with endogenous Stat5, shows sustained binding with tyrosine-phosphorylated betaIL3, and inhibits the tyrosine phosphorylation of endogenous Stat5. These results suggest that betaIL3 may have Stat5 docking sites, similar to those found in the EpoR, that facilitate the activation of Stat5 by Jak2 and raise the possibility that Epo may cross-activate or transmodulate the IL-3 receptor signaling pathways.
Chin
H
H
First Department of Internal Medicine and School of Allied Health Sciences, Tokyo Medical and Dental University, Bunkyoku, Japan.
Wakao
H
H
Miyajima
A
A
Kamiyama
R
R
Miyasaka
N
N
Miura
O
O
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D004921:Erythropoietin / Q000494:pharmacology*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D053614:Janus Kinase 2; D051379:Mice; D008894:Milk Proteins; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D016185:Receptors, Interleukin-3 / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects*; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D007377:Interleukin-3; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D016185:Receptors, Interleukin-3; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D004921:Erythropoietin; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2