Date of Award
Doctor of Philosophy (PhD)
Foxp3 is a transcription factor required for the development and function of regulatory T cells (Treg). Humans lacking functional Foxp3 are afflicted with uncontrolled systemic autoimmunity. How the Foxp3 protein is regulated post-translationally is unclear. Our previous studies demonstrate cyclin-dependent kinase 2 (CDK2) controls Foxp3+Treg function, but the mechanism by which this occurred was not identified. The CDKs are primarily thought to control cell cycle progression. However, recent studies suggest only CDK1 is required for normal mammalian cell cycle, raising questions about the biological role of the other CDKs. Specifically, mice genetically deficient in CDK2 are viable with no significant defects in cell cycle. We probed the Foxp3 sequence for the presence of CDK motifs, finding four such sites in the amino terminus. We confirmed that Foxp3 is phosphorylated by CDK2 using an in vitro kinase assay and mass spectrometry, as well as a phospho-specific antibody that recognizes one of the phosphorylated Foxp3-CDK motifs. We generated a mutant of Foxp3 lacking all four CDK motifs, which has increased half-life compared to wild-type Foxp3. CD4+ T cells transduced with a Foxp3-CDK motif mutant have increased function compared to cells transduced with wild-type Foxp3 as measured by induction and repression of canonical Foxp3 target genes, as well as the ability to suppress conventional T cell proliferation. These data suggest CDKs negatively regulate Foxp3 protein stability, which has an impact on Foxp3 function. To determine when the CDK cascade was actively regulating Foxp3 in vivo we investigated the role of the CDK2 inhibitor, p27kip1. Recent data shows TGFβ signaling drives expression of p27kip1 in B cells. TGFβ is also required for extrathymic induction of Foxp3 in conventional CD4+ T cells. We show that conventional T cells, which have high CDK2 activity and minimal p27kip1 expression, induce large amounts of p27kip1 along with Foxp3 in the presence of TGFβ. Additionally, T cells lacking p27kip1 have defective TGFβ-dependent Foxp3 induction. We hypothesize that TGFβ signaling is required to activate p27kip1 and stabilize Foxp3 protein levels in developing iTreg by repressing CDK2.
Morawski, Peter A., "Cyclin-Dependent Kinase 2 Regulates Foxp3 and Regulatory T Cell Function" (2013). Publicly Accessible Penn Dissertations. 900.