Mutagenesis of Erythropoietin Receptor Cytoplasmic Lysines Uncouples Erythropoietin-Dependent Growth From Downstream Signal Transduction Cascades.

Abstract 3611

Poster Board III-547

Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis, stimulating growth, preventing apoptosis, and promoting differentiation of red blood cell progenitors. Fundamental to this action is the ability of EPO to bind to its cognate receptor on the cell surface, the EPO receptor (EPO-R), and activate the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion.

Erythrocyte production relies heavily on precise control of EPO-mediated cell signaling cascades. The availability of EPO-R to EPO and the activation of subsequent signaling cascades are tightly regulated by the transit of mature EPO-R to the cell surface from the Golgi, and the mechanisms by which the receptor is internalized and down-regulated. Thus, small changes in plasma membrane EPO-R levels may be reflected in the intensity and duration of downstream signaling protein activation.

EPO-R is rapidly ubiquitinated and down-regulated from the cell surface upon EPO stimulation. EPO-R ubiquitination relies on more than one E3 ubiquitin ligase, the U-box containing E3 ubiquitin ligase, p33^Rul, and the RING finger E3 ubiquitin ligase SCF^βTrcp. The discovery that EPO-R is ubiquitinated and degraded by the proteasome and lysosome lead us to examine the role of EPO-R ubiquitination on cell signaling and proliferation. We hypothesized that EPO-R ubiquitination would result in down-regulation of EPO-mediated signaling cascades. This study characterized which lysines determined EPO-R ubiquitination, and assessed the ability of EPO-R lysine mutants to support EPO-mediated signaling and proliferation.

Surprisingly, substitution of all EPO-R cytoplasmic lysines (EPO-R K5R) abolished the ability of EPO-R to support EPO-mediated proliferation in BaF3 cells. However, EPO-dependent phosphorylation of EPO-R, AKT, PKB and STAT5 was detected, albeit at reduced levels. While ubiquitination defects commonly prolong receptor signaling, the kinetics of EPO-R K5R-mediated signaling pathways paralleled Ba/F3-EPO-R cells. Mutation of membrane proximal lysines, K256 and K276, resulted in decreased JAK2 phosphorylation and EPO-R phosphorylation and inhibited proliferation at 0.5U/ml EPO. However, the interaction between JAK2 and EPO-R was not affected. In addition, any EPO-R cytoplasmic lysine could support signaling and proliferation at physiologically elevated EPO concentrations demonstrating significant redundancy. The requirement of EPO-R cytoplasmic lysines to promote CFU-E formation is currently being evaluated by introduction of EPO-R mutants into fetal liver cells derived from EPO-R null mice. These results suggest that the EPO-R cytoplasmic lysines play a critical role in transmitting EPO-dependent growth signals within the cell.