A kinase-dead knock-in mutation in mTOR leads to early embryonic lethality and is dispensable for the immune system in heterozygous mice.
BACKGROUND
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. 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.


RESULTS
To 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.


CONCLUSIONS
Altogether, 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.
Shor
Boris
B
Inflammation Research Team, Drug Discovery, Johnson and Johnson Pharmaceutical Research and Development, LLC, Raritan, NJ, USA. shorb@wyeth.com
Cavender
Druie
D
Harris
Crafford
C
D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D047108:Embryonic Development / Q000235:genetics / Q000276:immunology*; D055879:Gene Knock-In Techniques; D006579:Heterozygote; D007107:Immune System / Q000196:embryology / Q000254:growth & development / Q000378:metabolism* / Q000473:pathology; D008213:Lymphocyte Activation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D051379:Mice; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000276:immunology / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D020123:Sirolimus / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A / Q000037:antagonists & inhibitors
D002352:Carrier Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A; C546843:mTOR protein, mouse