Exploring the Function of Sestrin/Gator As Novel Regulators of Hematopoiesis

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that responds to multiple environmental cues such as reactive oxygen species (ROS) and thereby regulates many fundamental biological processes including cell growth and autophagy. mTOR is found in two distinct multiprotein complexes, mTORC1 and mTORC2, of which mTORC1 has been established to play an important role in the regulation of hematopoiesis. For example, mTORC1 inhibition, combined with activation of canonical Wnt-signaling, was shown to increase long term repopulating (LT)-HSC self-renewal, whereas its activation depletes LT-HSCs.

The activity of mTORC1 is tightly controlled by multiple layers of upstream regulators, including the recently discovered GTPase activating protein (GAP) activity towards Rags (GATOR) complex, which is an AMP-Kinase/Tuberous sclerosis complex (TSC)-independent mTORC1 inhibitor induced by amino acid deprivation. GATOR consists of two subcomplexes, GATOR1 and GATOR2, whereby GATOR2 inhibits GATOR1. Inactivation of GATOR2 prevents mTORC1 activation by amino acids, whereas inactivation of GATOR1 constitutively activates mTORC1.

Sestrins (Sesn1, Sesn2 and Sesn3) are a family stress-inducible, redox-sensitive proteins that are involved in cellular- or organism-level adaptation to diverse metabolic challenges. They have been identified as direct interactors of GATOR2 and shown to inhibit mTORC1 by preventing GATOR2 from inhibiting GATOR1 in presence of amino acids. In quantitative affinity purification-mass spectrometry (AP-MS) and coimmunoprecipitation experiments with HeLa- and mouse embryonic stem cells harboring in situ GFP-tagged Sesn2, WDR59 and NPRL3 alleles, we could confirm the Sesn2/GATOR interaction under nearly physiological conditions.

To analyze the function of Sestrin/GATOR during hematopoiesis in more detail, we isolated Lin- Sca+ hematopoietic cells from the bone marrow of Sesn2^-/- mice and performed serial replating experiments and competitive hematopoietic repopulation experiments in lethally irradiated mice and observed that Sesn2^-/- progenitor cells proliferate significantly faster than their wild type counterparts albeit only during the initial engraftment phase. At later stages, the wild type cells took over, exceeding the Sesn2^-/- cells by 3-4 fold in peripheral blood, bone marrow and spleen three months after transplantation. This suggests that the increased proliferative potential of Sesn2^-/- progenitor cells leads to a depletion of the LT-HSC pool, strikingly resembling the phenotype of activated mTORC1.