SUMMARY Regulatory T (Treg) cell infiltration constitutes a prominent feature of pancreatic ductal adenocarcinoma (PDA). However, the immunomodulatory function of Treg cells in PDA is poorly understood. Here, we demonstrate that Treg cell ablation is sufficient to evoke effective anti-tumor immune response in early and advanced pancreatic tumorigenesis in mice. This response is dependent on IFN-γ producing cytotoxic CD8+ T cells. We show that Treg cells engage in extended interactions with tumor-associated CD11c+ dendritic cells (DCs) and restrain their immunogenic function by suppressing the expression of costimulatory ligands necessary for CD8+ T cell activation. Consequently, tumor-associated CD8+ T cells fail to display effector activities when Treg cell ablation is combined with DC depletion. We propose that tumor-infiltrating Treg cells can promote immune-tolerance by suppressing tumor-associated DC immunogenicity. The therapeutic manipulation of this axis might provide an effective approach for the targeting of PDA.
Members of the RAS small GTPase family regulate cellular responses to extracellular stimuli by mediating the flux through downstream signal transduction cascades. RAS activity is strongly dependent on its subcellular localization and its nucleotide-binding status, both of which are modulated by posttranslational modification. We have determined that RAS is posttranslationally acetylated on lysine 104. Molecular dynamics simulations suggested that this modification affects the conformational stability of the Switch II domain, which is critical for the ability of RAS to interact with guanine nucleotide exchange factors. Consistent with this model, an acetylationmimetic mutation in K-RAS4B suppressed guanine nucleotide exchange factor-induced nucleotide exchange and inhibited in vitro transforming activity. These data suggest that lysine acetylation is a negative regulatory modification on RAS. Because mutations in RAS family members are extremely common in cancer, modulation of RAS acetylation may constitute a therapeutic approach. M embers of the rat sarcoma (RAS) family of small monomeric GTPases function as molecular binary switches, with their biological activities determined by their nucleotide-binding state. When bound to GTP, RAS proteins engage a variety of downstream "effector" pathways to influence cellular behavior (1). As such, the nucleotide-binding state of RAS must be highly regulated in a cell, and this regulation is accomplished through the activity of positive and negative cofactors. Wild-type RAS has low intrinsic GTPase activity and thus relies on GTPaseactivating proteins (GAPs) to hydrolyze GTP efficiently. Guanine nucleotide exchange factors (GEFs) facilitate the reloading of GDP-bound RAS with GTP. Activating-point mutations in RAS proteins are common in cancer, with missense mutations at codons 12 and 13 being the most prevalent (2). These particular mutations affect the endogenous enzymatic activity of RAS, but have a much greater effect on GAP-induced GTP hydrolysis, effectively shifting the nucleotide-binding equilibrium of RAS toward its constitutively active (i.e., GTP-bound) state (3).The nucleotide-binding state affects RAS activity by influencing its 3D structure. When RAS binds to GTP, it undergoes a conformational change that primarily affects two regions of the protein: Switch I, which binds to effectors and GAPs, and Switch II, which is critical for GEF and GAP activity and for interaction with PI3K (4-7). Mutations that impinge on the nucleotide-dependent conformation change affect the ability of RAS to release nucleotide in the presence of GEF and to activate downstream effectors (8). In essence, proper RAS function requires the ability to cycle between its active and inactive conformations.Within a cell, RAS proteins must associate with cellular membranes to transmit signals to downstream effector proteins. Because RAS itself is not a transmembrane protein, its proper localization is accomplished through posttranslational lipidation, primarily by irreversible farnesylation o...
Rho GTPases are critical for mitosis progression and completion of cytokinesis. During mitosis, the GDP/GTP cycle of Rho GTPases is regulated by the exchange factor Ect2 and the GTPase activating protein MgcRacGAP which associates with the kinesin MKLP1 in the centralspindlin complex. We report here that expression of Ect2, MgcRacGAP, and MKLP1 is tightly regulated during cell cycle progression. These three genes share similar cell cycle-related signatures within their promoter regions: (i) cell cycle gene homology region (CHR) sites located at ؊20 to ؉40 nucleotides of their transcription start sites that are required for repression in G 1 , (ii) E2F binding elements, and (iii) tandem repeats of target sequences for the CUX1 transcription factor. CUX1 and E2F1 bind these three promoters upon S-phase entry, as demonstrated by chromatin immunoprecipitation, and regulate transcription of these genes, as established using promoterluciferase reporter constructs and expression of activated or dominant negative transcription factors. Overexpression of either E2F1 or CUX1 increased the levels of the endogenous proteins whereas small interfering RNA knockdown of E2F1 or use of a dominant negative E2F1 reduced their expression levels. Thus, CUX1, E2F, and CHR elements provide the transcriptional controls that coordinate induction of Ect2, MgcRacGAP, and MKLP1 in S phase, leading to peak expression of these interacting proteins in G 2 /M, at the time they are required to regulate cytokinesis.
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