Numb-dependent integration of pre-TCR and p53 function in T-cell precursor development

Numb asymmetrically segregates at mitosis to control cell fate choices during development. Numb inheritance specifies progenitor over differentiated cell fates, and, paradoxically, also promotes neuronal differentiation, thus indicating that the role of Numb may change during development. Here we report that Numb nuclear localization is restricted to early thymocyte precursors, whereas timed appearance of pre-T-cell receptor (pre-TCR) and activation of protein kinase Cθ promote phosphorylation-dependent Numb nuclear exclusion. Notably, nuclear localization of Numb in early thymocyte precursors favors p53 nuclear stabilization, whereas pre-TCR-dependent Numb nuclear exclusion promotes the p53 downmodulation essential for further differentiation. Accordingly, the persistence of Numb in the nucleus impairs the differentiation and promotes precursor cell death. This study reveals a novel regulatory mechanism for Numb function based on its nucleus–cytosol shuttling, coupling the different roles of Numb with different stages of T-cell development.Cell fate decision of dividing progenitor-derived cells is a crucial event in development and diseases. Cell fate is often regulated by asymmetric cell division, which is a process by which progenitors asymmetrically segregate certain cell fate determinants during division, to generate two functionally different cells.^1,2 The adaptor protein Numb was initially identified in Drosophila as a critical cell fate determinant,³ where loss of Numb and its homolog Numb-like results in the loss of neural progenitors, indicating that the presence of Numb is essential for maintaining the progenitors during the initial progenitor versus neural fate decision.^4,5 However, re-expression of Numb is also required for further neural differentiation,^6,7 indicating that the role of Numb in the same tissue may change over time.Numb function in the immune system has been partially explored.^8,9 Numb is involved in asymmetric division in hematopoietic stem cells,¹⁰ thymocytes¹¹ and mature T lymphocytes.^12,13 T cells develop from intrathymic CD4⁻CD8⁻ double-negative (DN) precursors that, after progression through DN1 (CD44⁺CD25⁻), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁺) and DN4 (CD44⁻CD25⁻), have to decide between proliferation, to increase the total number of precursors, or differentiation into CD4⁺CD8⁺ double-positive (DP) cells. This decision is made during DN3 stage and appears to be dependent on asymmetric segregation of Numb.¹¹As Numb is a well-characterized inhibitor of Notch-1 receptor signaling pathway,¹⁴ the ability of Numb to regulate cell fate decisions during development has been associated with this Numb function.¹⁵ However, the role of Numb during development could not be restricted to the control of Notch-1 signaling, as Numb has been implicated in the regulation of a variety of biochemical pathways, including the tumor suppressor p53.¹⁶ Increasing evidence suggests that p53 regulates cell differentiation in addition to cell proliferation, apoptosis and senescence.^17,18Notably, T-cell development is regulated by both Notch-1 and p53. Notch-1 signals appear to be critical for the very early steps of T-cell development (i.e. T-cell commitment).¹⁹ The involvement of p53 has been instead reported in the transition from the DN to the DP stage. However, while the overexpression of p53 during DN3 stage promotes a block in the differentiation and proliferation, resulting in a small thymus size,^20,21 loss of p53 apparently does not affect thymocyte development, even though the vast majority of spontaneous malignancies in p53^−/− mice are lymphomas.²² Thus, the double function of Numb could be dependent on two different pathways, which may be differentially triggered during selected differentiation stages.Recent data describe the presence of Numb in the nuclear compartment,²³ besides its known cytoplasmic localization, raising the possibility that different Numb functions could be regulated by its differential subcellular localization. However, whether Numb may have different subcellular localizations in precursors or more differentiated T cell, how Numb import is regulated or how the nuclear localization affects its function during T-cell development remain unexplored. Here we show that Numb is an important regulator of p53 pathway during T-cell development, and we describe a novel molecular mechanism involved in the differential regulation of Numb–p53 axis based on the regulation of Numb nuclear import, emerging an interesting scenario where Numb can act as a regulator of two fundamental pathways during T-cell development.