Notch1 intracellular domain increases cytoplasmic EZH2 levels during early megakaryopoiesis.
Notch pathway is a well-known factor in the development of lymphoid lineage. However, its role in the myeloid lineage has remained ambiguous. We looked into the effect of Notch1 on the megakaryocytic lineage commitment and found an increase in megakaryocyte-specific lineage markers upon transfection with Notch1 intracellular domain (NICD). This effect was mediated by Akt whereby constitutive activation of Akt increased the megakaryocyte markers, whereas inhibition of Akt signalling reduced these marker levels. Along with the change in differentiation status, NICD-induced initiation of early megakaryopoiesis was accompanied by an increased cytoplasmic enhancer of zeste homolog-2 (EZH2) expression. This process was found to be Akt-dependent, and inhibition or overexpression of Akt lead to concurrent changes in EZH2 levels. To elucidate the function of EZH2 in the cytoplasm, novel cytoplasmic interactors of EZH2 were identified by co-immunoprecipitation followed by matrix-assisted laser desorption ionization MS/MS-based protein identification, and thus, PDIA1 and LIM domain kinase-1 (LIMK1) were identified. Interaction of EZH2 with LIMK1 changed the activity of cofilin (a downstream target of LIMK1) towards actin filaments, thereby leading to lower filamentous actin content within these cells. Thus, Notch1 not only induces early megakaryopoiesis but also prepares these cells for subsequent morphological changes.
Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, West Bengal, India.
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Roy
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Basak
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Banerjee
D051339:Actin Depolymerizing Factors / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000071221:Enhancer of Zeste Homolog 2 Protein; D006801:Humans; D020014:K562 Cells; D054461:Lim Kinases / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology*; D063151:Polycomb Repressive Complex 2; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051881:Receptor, Notch1 / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015854:Up-Regulation
D051339:Actin Depolymerizing Factors; D004268:DNA-Binding Proteins; D051881:Receptor, Notch1; D014157:Transcription Factors; D011393:Procollagen-Proline Dioxygenase; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2; C515892:LIMK1 protein, human; D054461:Lim Kinases; D051057:Proto-Oncogene Proteins c-akt; C526497:P4HB protein, human; D019704:Protein Disulfide-Isomerases