AURKA Kinase Controls Erythroblast Enucleation Via Regulation of Centrosome Localization and ECT2 Degradation

Mature red blood cells (RBCs) in the peripheral circulation of mammals are nuclear-free. Erythroblast enucleation is proposed to be analogous to asymmetric cell division based on cellular progress. the molecular mechanisms of how spherical erythroblasts determine the direction of nuclear polarization and accomplish the final separation of the nucleus from the nascent reticulocytes remain largely unclear, until now.

Aurora kinases are highly conserved serine/threonine kinases required for regulation of mitosis (AURKA and AURKB). However, up to now, the role of Aurora kinases in erythroid cells, especially in erythroblast enucleation has been fully undefined.

Here, we found that AURKA and AURKB messenger RNA (mRNA) expression levels were similar early in terminal differentiation, but followed by a prominent increase in AURKA levels in polychromatic and orthochromatic erythroblasts, whereas AURKB levels were dramatically downregulated. The expression patterns of Auraro kinases were further confirmed by western blotting analysis. More notably, the expression of Aurora kinases is low or absent in most adult tissues or cells due to their lower proliferation rates. However, AURKA and AURKB are still highly expressed in orthochromatic erythroblasts compared to other precursor erythroblasts. The unconventional expression profile of Aurora kinases, especially AURKA, during terminal erythroid differentiation suggests an extraordinary role in erythroblast enucleation.

To explore the role of Aurora kinases in erythroblast enucleation, we first tested whether pharmacological inhibition of these kinases blocked the enucleation of human erythroblasts. Treatment of erythroblasts with MLN8237 (Aliseritib, AURKA selective inhibitor), AZD1152(Barasertib, AURKB selective inhibitor), or AT9283 (AURKA and AURKB inhibitor) dramatically blocked enucleation in a dose-dependent manner. Furthermore, we found that knockdown of Aurora kinases by siRNA indeed resulted in impaired enucleation. We next examined whether Aurora kinases regulated nuclear polarization, which is considered the onset of erythroblast enucleation, by measuring the △centroid distribution (distance between centers of the cytoplasm and nucleus). We found that erythroblast nuclear polarization is AURKA dependent, but not AURKB dependent.

Intriguingly, high-resolution confocal microscopy analyses revealed that AURKA colocalized with the centrosome on the side of the nucleus opposite its membrane point of contact during polarization and subsequently solely translocated to the anterior end of the protrusive nucleus upon nuclear exit, whereas AURKB consistently localized to the actin contraction ring during enucleation.

Colocalization of AURKA and centrosome during nuclear polarization raised the question whether AURKA controls polarization by regulating centrosome organization and maturation. Co-immunoprecipitation (co-IP) verified a direct interaction between AURKA and γ-tubulin. We found that AURKA loss did not alter the expression levels of γ-tubulin in erythroblasts. However, immunofluorescence showed that MLN8237 treatment obviously impaired the proper organization of centrosome. These phenotypes of centrinone-B, an inhibitor of the master centriole biogenesis regulator PLK4 treated erythroblasts mimic the results of AUKRA-deficient erythroblasts, suggesting a close connection between AUKRA and centrosome in polarization during erythroblast enucleation.

We intriguingly found that ECT2 (Epithelial Cell Transforming 2), was only constricted at the rear of the thanslocating nucleus in enucleating erythroblasts. Inhibition of AURKA obviously impaired this transformation, exhibiting persistent cytoplasmic localization. MLN8237 treatment resulted in a significant increase of ECT2 proteins in K562 cells.In contrast to control scramble siRNA, ECT2 siRNA significantly increased the enucleation of MLN8237-treated erythroblast.

The translocation of AURKA, which is originally restricted to centrosome relied on γ-tubulin interaction during nuclear polarization, was to directly degrade ECT2 at the anterior end of the protrusive nucleus for final nuclear expulsion. Our findings reveal a previously unrecognized localization and role of Aurora kinases in terminal erythroid differentiation and provide new mechanistic insights into erythroblast enucleation.