Abstract
BackgroundThe mammalian target of rapamycin protein (mTOR) is an evolutionarily conserved kinase that regulates protein synthesis, cell cycle progression and proliferation in response to various environmental cues. As a critical downstream mediator of PI3K signaling, mTOR is important for lymphocyte development and function of mature T and B-cells. Most studies of mTOR in immune responses have relied on the use of pharmacological inhibitors, such as rapamycin. Rapamycin-FKBP12 complex exerts its immunosuppressive and anti-proliferative effect by binding outside the kinase domain of mTOR, and subsequently inhibiting downstream mTOR signaling.ResultsTo determine the requirement for mTOR kinase activity in the immune system function, we generated knock-in mice carrying a mutation (D2338) in the catalytic domain of mTOR. While homozygous mTOR kd/kd embryos died before embryonic day 6.5, heterozygous mTOR+/kd mice appeared entirely normal and are fertile. mTOR +/kd mice exhibited normal T and B cell development and unaltered proliferative responses of splenocytes to IL-2 and TCR/CD28. In addition, heterozygousity for the mTOR kinase-dead allele did not sensitize T cells to rapamycin in a CD3-mediated proliferation assay. Unexpectedly, mTOR kinase activity towards its substrate 4E-BP1 was not decreased in hearts and livers from heterozygous animals.ConclusionAltogether, our findings indicate that mTOR kinase activity is indispensable for the early development of mouse embryos. Moreover, a single wild type mTOR allele is sufficient to maintain normal postnatal growth and lymphocyte development and proliferation.
Highlights
The mammalian target of rapamycin protein is an evolutionarily conserved kinase that regulates protein synthesis, cell cycle progression and proliferation in response to various environmental cues
Generation of mammalian target of rapamycin protein (mTOR) D2338A knock-in mice To understand the in vivo role of the mTOR kinase catalytic activity, we set out to generate knock-in mice with a targeted replacement of the wild type mTOR allele with a mutant version in which Asp 2338 was changed to Ala (D2338A) (Fig 1, Methods)
The proliferative response of T cells to anti-CD3 stimulation was well inhibited by rapamycin with the half maximal inhibitory concentration (IC50) values of ~1 nM for cells of both genotypes (Fig. 8). These results indicate that T cell proliferation is normal in mTOR+/kd mouse and that a heterozygousity for a kinasedead mTOR does not sensitize T cells to rapamycin in vitro
Summary
The mammalian target of rapamycin protein (mTOR) is an evolutionarily conserved kinase that regulates protein synthesis, cell cycle progression and proliferation in response to various environmental cues. As a critical downstream mediator of PI3K signaling, mTOR is important for lymphocyte development and function of mature T and B-cells. MTOR acts as a sensor kinase that coordinates cellular response to growth factors, nutrients and energy availability in mammalian cells [1,2]. Deregulation of mTOR signaling, which is caused by the loss of critical tumor suppressors (PTEN, TSC1/2, LKB1), somatic mutations or gene amplifications of PI3CA (p110 alpha subunit of PI3K) or activating mutations in AKT, leads to increased cell growth, cell survival, and suppression of autophagy [5]
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