“RHEX-26”, a Novel EPO/EPOR-Regulated Mediator of Human Erythroid Progenitor Cell Expansion

Abstract 682

Erythropoietin (EPO) and its receptor (EPOR) serve well as an informative paradigm for understanding hematopoietic growth factor effects and receptor action mechanisms. Recently, interest in EPO/EPOR investigations has been heightened based on the clinical emergence of novel EPO mimetics, and by reported EPO/EPOR activities in modulating cellular immunity, diabetic susceptibility, retinopathy and the cytoprotection of select non-hematopoietic tissues. To better understand EPOR action modes, we presently have applied a global phosphoproteomics approach to specifically discover new targets of EPO/EPOR-regulated tyrosine phosphorylation. Using an EPO-dependent erythroid progenitor cell model, our LC-MS/MS approach was first validated for known EPO/EPOR-targets (e.g., JAK2, STATs, SHIPs, PLCgamma, GABs). Second, among certain known targets, new phosphorylation sites were identified. Third, several novel major downstream targets of EPO/EPOR were discovered. One (a previously uncharacterized open reading frame here designated as “RHEX26”) was induced ∼20-fold in tyrosine-phosphorylation by EPO treatment. Quantitative RT-PCR analyses of tissues and primary hematopoietic cells indicate RHEX26 expression to be erythroid- restricted. Antibodies prepared to RHEX26 and tyrosine phosphorylated-RHEX26 confirmed strong EPO-induced phosphorylation; cell fractionation and Western blot studies demonstrated membrane residence of this 26 kDa phosphoprotein; and immunofluorescence microscopy showed sharp RHEX26 sub-localization as an integral plasma membrane protein. In functional analyses, lentiviral shRNA knockdown of RHEX26 proved to markedly inhibit the EPO-dependent expansion of erythroid progenitor cells (clonal colony-forming analyses demonstrated >80% specific inhibition), while effects on cell viability were limited. Mechanistically, RHEX26 is coupled (in part) to GRB2, with co-immunoprecipitation observed specifically upon EPO exposure, but without dependency on RHEX26 tyrosine phosphorylation. Finally, genealogy analyses intriguingly indicate RHEX26 to be well-conserved only in H. sapiens and primates, while no ortholog is present in the rat, mouse, or zebrafish. RHEX26, as a novel EPO/EPOR 26 kDa target, therefore is proposed to comprise an important new Regulator of Human Erythroid eXpansion.