Roles for an Epo Receptor Tyr-343 Stat5 Pathway in Proliferative Co-signaling with Kit

Ke Li,Chris Miller,Shailajia Hegde,Don Wojchowski

doi:10.1074/jbc.m307182200

Abstract

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.

Highlights

Erythropoiesis depends sharply upon combined sets of signals that are relayed by the single transmembrane receptors for erythropoietin (EpoR),1 and stem cell factor (Kit)
Compared first in FDCW2 cells were the activities of wild-type (EE-WT), Tyr(P)-343-retaining (EE-T-Y343), and two Tyr(P)-null Epo receptor (EpoR) forms (EE-F8 and EE-T-Y343F)
The present investigations sought to quantitatively assess relative contributions of select EpoR Tyr(P) sites to proliferative signaling independently and in the presence of Kit co-activation. Aspects of these studies selected for discussion include: 1) the importance of attention paid to matched receptor expression levels; 2) the significant bolstering of Tyr(P)-null EpoR form activity by Tyr(P)-343, Tyr(P)479, and to an extent, Tyr(P)-464-coupled pathways; 3) apparent selective roles for an EpoR Tyr(P)-343 Stat5 binding site in Kit co-signaling; and 4) possible molecular mechanisms that underlie EpoR and Kit co-signaling

Summary

Introduction

Erythropoiesis depends sharply upon combined sets of signals that are relayed by the single transmembrane receptors for erythropoietin (EpoR), and stem cell factor (Kit). A series of EpoR Tyr(P) mutants were prepared as hEGFR-EpoR “EE” chimeras and were expressed using MSCV-based vectors in IL-3-dependent FDC cells [33], as well as ES cell-derived erythroid G1E-2 cells [34] In cells expressing these Epo receptor forms at matched physiological levels (as isolated by FACS), the abilities of these Tyr(P) mutant EE receptors to support progenitor cell proliferation were assayed in the absence or presence of endogenous Kit co-activation. This approach was prompted by signaling studies in several laboratories and systems that underline the importance of attention paid to EpoR expression levels [33, 35] and by the limited availability of useful antibodies to the activated cell sorter; PI3K, phosphatidylinositol 3-kinase; IMDM, Iscove’s modified Dulbecco’s medium; CFUe, colony-forming unit erythroid; h, human; m, murine. This co-signaling did not involve detectable effects of Kit on Stat activity and instead is proposed to involve specific interactions between

Methods

Results

Conclusion