CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

Erythroid enucleation is the final stage of erythroid terminal differentiation and involves the separation of an orthochromatic erythroblast into two daughter cells; a pyrenocyte containing the extruded nucleus, and the other a reticulocyte that will become a red blood cell. Our previous work identified CDK9 as a regulator of erythroid enucleation that appears to act independently of its known role in regulating RNA polymerase II transcription, suggesting the potential for a new CDK9 role. Using a co-immunoprecipitation and mass spectrometry approach, we have identified the interactome of CDK9 in differentiating erythroblasts. We show that CDK9 interacts with a RanGTP-NEMP1-Importin β complex during erythroid terminal differentiation, and inhibition of importin β in erythroblasts blocks erythroid enucleation. Using imaging analysis and functional assays of enucleating erythroblasts, we show that CDK9 and importin β co-locate at a critical site of activity opposite to the nucleus before nuclear extrusion and we describe a novel finding that physically links CDK9 and importin β activity prior to CaM/Ca ²⁺ signalling and subsequent F-actin activity to achieve enucleation.