Roles for an Epo receptor Tyr-343 Stat5 pathway in proliferative co-signaling with kit.
Erythroid progenitor cell expansion depends upon co-signaling by Epo receptor (EpoR) and Kit, but underlying mechanisms are incompletely understood. To quantitatively analyze EpoR contributions to co-signaling, phosphotyrosine (Tyr(P)) mutants were expressed as human epidermal growth factor (hEGF) receptor-mEpoR EE chimeras at matched and physiological levels in FDCW2 hematopoietic progenitor cells and were assayed for proliferative activities in the absence or presence of endogenous Kit stimulation. Two Tyr(P)-null (but Jak2-coupled) EpoR forms each retained <or=25% of the wild-type activity, whereas the add-back of single Tyr(P) sites in the EpoR forms EE-T-Y343 (Stat5 binding site), EE-Y479 (p85/phosphatidylinositol 3-kinase binding site), or EE-Y464 (Src kinase binding site) significantly enhanced activities (to 100, 95, and 50% of EE-WT (wild type) levels, respectively). EE-Y343&Y401 and EEF343&F401 double add-back and deletion constructs were also prepared and were shown to possess 90 and <or=50% of wild-type activity. In contrast, efficient Kit co-signaling activity was retained only by EE-T-Y343 and EE-Y343&Y401 EpoR forms. EE-T-Y343 together with EE-T-Y343F and EE-WT EpoR forms were also analyzed in embryonic stem cell-derived erythroid G1E-2 cells with highly comparable outcomes, including the ability of EE-T-Y343 (but not EE-T-Y-343F) to synergize with Kit. Despite specific connection of EE-T-Y343 to Stat5, the contributions of Kit to EpoR-dependent proliferation did not involve Kit effects on Stat5 activation (but was limited by the mutation of Kit Tyr(P)-567 and Tyr(P)-569 Src kinase recruitment sites). Instead, co-signaling appears to depend upon the downstream integration of Kit signals with the targets of an EpoR/Jak2/Y343/Stat 5 response axis.
Li
Ke
K
Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Miller
Chris
C
Hegde
Shailajia
S
Wojchowski
Don
D
D000818:Animals; D001665:Binding Sites; D002455:Cell Division; D002460:Cell Line; D002469:Cell Separation; D016207:Cytokines / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004921:Erythropoietin / Q000378:metabolism; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D051379:Mice; D008894:Milk Proteins; D008957:Models, Genetic; D009154:Mutation; D019000:Phosphotyrosine / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D017467:Receptors, Erythropoietin / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D016207:Cytokines; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D017467:Receptors, Erythropoietin; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D004921:Erythropoietin; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D019009:Proto-Oncogene Proteins c-kit