Mutagenesis of Erythropoietin Receptor Cytoplasmic Lysines Uncouples Erythropoietin-Dependent Growth From Downstream Signal Transduction Cascades.
Bibliographic record
Abstract
Abstract 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, p33Rul, and the RING finger E3 ubiquitin ligase SCFβTrcp. 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. Disclosures: No relevant conflicts of interest to declare.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.004 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".