Summary The differentiation and activation of T cells are critically modulated by MAP kinases, which are in turn feed-back regulated by dual-specificity phosphatases (DUSPs) to determine the duration and magnitude of MAP kinase activation. DUSP4 (also known as MKP2) is a MAP kinase-induced DUSP member that is dynamically expressed during thymocyte differentiation. We generated DUSP4-deficient mice to study the function of DUSP4 in T-cell development and activation. Our results showed that thymocyte differentiation and activation-induced MAP kinase phosphorylation were comparable between DUSP4-deficient and wild type mice. Interestingly, activated DUSP4−/− CD4 T cells were hyperproliferative while DUSP4−/− CD8 T cells proliferated normally. Further mechanistic studies suggested that the hyperproliferation of DUSP4−/− CD4 T cells resulted from enhanced CD25 expression and IL-2 signaling through increased STAT5 phosphorylation. Immunization of the DUSP4−/− mice recapitulated the T-cell hyperproliferation phenotype in antigen recall responses, while the profile of Th1/Th2-polarized antibody production was not altered. Combined, these results suggest that other DUSPs may compensate for DUSP4 deficiency in T-cell development, MAP kinase regulation, and Th1/Th2-mediated antibody responses. More importantly, our data indicate that DUSP4 suppress CD4 T-cell proliferation through novel regulations in STAT5 phosphorylation and IL-2 signaling.

Immune responses are critically regulated by the functions of CD4 helper T cells. Based on their secreted cytokines, helper T cells are further categorized into different subsets like Treg or Th17 cells, which suppress or promote inflammatory responses, respectively. Signals from IL-2 activate the transcription factor STAT5 to promote Treg but suppress Th17 cell differentiation. Our previous results found that the deficiency of a dual-specificity phosphatase, DUSP4, induced STAT5 hyper-activation, enhanced IL-2 signaling, and increased T cell proliferation. In this report, we examined the effects of DUSP4 deficiency on helper T cell differentiation and STAT5 regulation. Our in vivo data showed that DUSP4 mice were more resistant to the induction of autoimmune encephalitis, while in vitro differentiations revealed enhanced iTreg and reduced Th17 polarization in DUSP4-deficient T cells. To study the cause of this altered helper T cell polarization, we performed luciferase reporter assays and confirmed that, as predicted by our previous report, DUSP4 over-expression suppressed the transcription factor activity of STAT5. Surprisingly, we also found that DUSP4-deficient T but not B cells exhibited elevated STAT5 protein levels, and over-expressed DUSP4 destabilized STAT5 in vitro; moreover, this destabilization required the phosphatase activity of DUSP4, and was insensitive to MG132 treatment. Finally, domain-mapping results showed that both the substrate-interacting and the phosphatase domains of DUSP4 were required for its optimal interaction with STAT5, while the coiled-coil domain of STAT5 appeared to hinder this interaction. Our data thus provide the first genetic evidence that DUSP4 is important for helper T cell development. In addition, they also help uncover the novel, DUSP4-mediated regulation of STAT5 protein stability.

In this study we identified Pdc2, the fission yeast ortholog of human Pat1b protein, which forms a complex with Lsm1-7 and plays a role in coupling deadenylation and decapping. The involvement of Pdc2 in RNA degradation and P-body function was also determined. We found that Pdc2 interacts with Dcp2 and is required for decapping in vivo. Although not absolutely essential for P-body assembly, overexpression of Pdc2 enhanced P-body formation even in the absence of Pdc1, the fission yeast functional homolog of human Edc4 protein, indicating that Pdc2 also plays a role in P-body formation. Intriguingly, in the absence of Pdc2, Lsm1 was found to accumulate in the nucleus, suggesting that Pdc2 shuttling between nucleus and cytoplasm plays a role in decreasing the nuclear concentration of Lsm1 to increase Lsm1 in the cytoplasm. Furthermore, unlike other components of P-bodies, the deadenylase Ccr4 did not accumulate in P-bodies in cells growing under favorable conditions and was only recruited to P-bodies after deprivation of glucose in a Pdc2-Lsm1-dependent manner, indicating a function of Pdc2 in cellular response to environmental stress. In supporting this idea, mutants are defective in recovery from glucose starvation with a much longer time to re-enter the cell cycle. In keeping with the notion that Pat1 is a nucleocytoplasmic protein, functioning also in the nucleus, we found that Pdc2 physically and genetically interacts with the nuclear 5'-3' exonuclease Dhp1. A function of Pdc2-Lsm1, in concert with Dhp1, regulating RNA by promoting its decapping/destruction in the nucleus was suggested.

BackgroundProtein phosphates 4 (PP4), encoded by the ppp4c gene, is a ubiquitously expressed phosphatase that has been implicated in the regulation of cytokine signaling and lymphocyte survival; recent reports suggest that PP4 may be involved in pre-TCR signaling and B cell development. However, whether PP4 also modulates the functions of peripheral T cells has not been investigated due to the lack of a suitable in vivo model. Treg cells are a specialized subset of CD4 helper T cells that can suppress the proliferation of activated effector T cells. In the absence of this negative regulation, autoimmune syndromes and inflammatory diseases, such as human Crohn’s disease, will arise.ResultsIn this report, we generated mice with T cell-specific ablation of the ppp4c gene (CD4cre:PP4f/f) and a Foxp3-GFP reporter gene to examine the roles of PP4 in Treg development and function. Characterizations of the CD4cre:PP4f/f mice showed that PP4 deficiency induced partial αβ T lymphopenia and T cell hypo-proliferation. Further analyses revealed significant reductions in the numbers of thymic and peripheral Treg cells, as well as in the efficiency of in vitro Treg polarization. In addition, PP4-deficient Treg cells exhibited reduced suppressor functions that were associated with decreased IL-10, CTLA4, GITR and CD103 expression. More interestingly, the CD4cre:PP4f/f mice developed spontaneous rectal prolapse and colitis with symptoms similar to human Crohn’s disease. The pathogenesis of colitis required the presence of commensal bacteria, and was correlated with reduced Treg cells in the gut. Nevertheless, PP4-deficient Treg cells were still capable of suppressing experimental colitis, suggesting that multiple factors contributed to the onset of the spontaneous colitis.ConclusionsWhile the molecular mechanisms remain to be investigated, our results clearly show that PP4 plays a non-redundant role for the differentiation, suppressor activity and gut homeostasis of Treg cells. The onset of spontaneous colitis in the CD4cre:PP4f/f mice further suggests that PP4 is essential for the maintenance of protective gut immunity. The CD4cre:PP4f/f mice thus may serve as a good model for studying the interactions between Treg cells and gut commensal bacteria for the regulation of mucosal immunity.

Stress granules (SGs) are cytoplasmic aggregates formed upon stress when untranslated messenger ribonucleoproteins accumulate in the cells. In a green fluorescent protein library screening of the fission yeast SG proteins, Puf2 of the PUF family of RNA-binding proteins was identified that is required for SG formation after deprivation of glucose. Accordingly, the puf2 mutant is defective in recovery from glucose starvation with a much longer lag to reenter the cell cycle. In keeping with these results, Puf2 contains several low-complexity and intrinsically disordered protein regions with a tendency to form aggregates and, when overexpressed, it represses translation to induce aggregation of poly(A) binding protein Pabp, the signature constituent of SGs. Intriguingly, overexpression of Puf2 also enhances the structure of processing bodies (PBs), another type of cytoplasmic RNA granule, a complex of factors involved in mRNA degradation. In this study, we demonstrate a function of the fission yeast PB in SG formation and show Puf2 may provide a link between these two structures.

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The clonal expansion of activated T cells is pivotal for the induction of protective immunity. Protein phosphatase 4 (PP4) is a ubiquitously expressed serine/threonine phosphatase with reported functions in thymocyte development and DNA damage responses. However, the role of PP4 in T cell immunity has not been thoroughly investigated. In this report, our data showed that T cell-specific ablation of PP4 resulted in defective adaptive immunity, impaired T cell homeostatic expansion, and inefficient T cell proliferation. This hypo-proliferation was associated with a partial G1-S cell cycle arrest, enhanced transcriptions of CDK inhibitors and elevated activation of AMPK. In addition, resveratrol, a known AMPK activator, induced similar G1-S arrests, while lentivirally-transduced WT or constitutively-active AMPKa1 retarded the proliferation of WT T cells. Further investigations showed that PP4 co-immunoprecipitated with AMPKa1, and the over-expression of PP4 inhibited AMPK phosphorylation, thereby implicating PP4 for the negative regulation of AMPK. In summary, our results indicate that PP4 is an essential modulator for T cell proliferation and immune responses; they further suggest a potential link between PP4 functions, AMPK activation and G1-S arrest in activated T cells.

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5′ splice-site (5′SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28’s ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.