<p>Multiple signaling pathways control protein synthesis by modulating translation initiation factors. Map Kinase Integrating Kinase 1 (Mnk1) relays signals to its major downstream target eIF4E. Activation of Mnk1 and subsequent phosphorylation of eIF4E results in changes in translation rates for subsets of mRNAs. Both the Erk1/2 and p38 MAPK pathways activate Mnk1 meaning that Mnk1 responds to growth signals through Erk1/2 and stress signals through p38 MAPK. However, it is not clear how Mnk1 mediates translational changes specific to each pathway. We investigated the activation of Mnk1 by stress and cytokines through the p38 MAPK pathway. We found that of the four different p38 MAPK isoforms, p38&alpha; alone controls acute stress and cytokine signaling to translation machinery. Furthermore, this regulatory axis is greatly diminished in neurons. We discovered that p38&alpha; expression is repressed in the brain due to two neuron-selective microRNAs, miR-124 and -128. Next, we investigated the mechanism of p38&alpha; mediated Mnk1 activation to see if it differed from Erk1/2 mediated activation. Looking at the induced binding of Mnk1 to eIF4G, we found that the dissociation rate varies depending on the activating pathways. This shows that Mnk1 is not a true convergence point of p38 and Erk1/2 MAPK pathways resulting in identical downstream effects, but that Mnk1 mediates pathway specific effects on translation factors.</p> / Dissertation

<p>Diacylglycerol (DAG), a lipid messenger generated upon T cell receptor (TCR) engagement, mediates signaling through the IKK/NF-&#954;B and Ras/ERK pathways. Further downstream of the Ras/ERK pathway are mammalian target of rapamycin (mTOR) and MAP kinase signal integrating kinases Mnk1 and Mnk2. While mTOR acts as a critical regulator of T cell metabolism, homeostasis and function, Mnk1 and Mnk2 phosphorylate the initiation factor eIF4E that plays an important role in cap-dependent mRNA translation. Diacylglycerol kinases (DGKs) terminate DAG-mediated signals by phosphorylating DAG into phosphatidic acid. T cells that lack both &#945; and &#950; isoforms of DGK accumulate excess DAG upon activation, resulting in hyper-activation of the IKK/NF-&#954;B, Ras/ERK and mTOR pathways, hypersensitivity to TCR stimulation, and loss of self-tolerance. Here, we have examined the mechanisms by which dysregulated DAG-mediated signaling affects T cell function. To this end, we studied the effects of hyper-activating individual DAG-mediated pathways (IKK/NF-&#954;B and TSC/mTOR) on T cell function. We also examined the role of ERK-activated kinases Mnk1 and Mnk2 in T cell function.</p><p>Using mice with T cell-specific expression of a constitutively active form of IKK&#946; (`IKK' mice), we found that uncontrolled IKK&#946;/NF-&#954;B signaling promotes T cell apoptosis and attenuates responsiveness to TCR stimulation. Defective IL-2 production and increased FasL expression contributed to enhanced IKK T cell apoptosis. Impaired IKK T cell activation and proliferation were associated with defects in TCR signaling, and upregulation of the cell surface inhibitory receptor PD1. In vivo, IKK T cells mounted a compromised antigen-specific CD8 T cell response with curtailed expansion and exaggerated contraction phases. Notably, expression of transcriptional repressor Blimp1 (a regulator of T cell exhaustion) was increased in IKK T cells, and conditionally deleting Blimp1 was able to largely restore responsiveness to TCR stimulation.</p><p>Investigating Mnk1/2 double knockout (DKO) mice, we found that Mnk1 and Mnk2 are dispensable for T cell development and function, but important for the pathogenesis of experimental autoimmune encephalomyelitis (EAE). TCR engagement activated Mnk1/2 in a Ras/ERK-dependent manner in primary T cells, and was inhibited by DGK &#945; and &#950;. Mnk1/2 deficiency did not affect the development of conventional &#945;&#946; T cells, regulatory T cells, or invariant NKT cells. Mature T cells from DKO mice showed normal activation and CD4 TH differentiation ex vivo, but DKO mice developed lower clinical scores than WT counterparts in an EAE model, correlating with a smaller pool of MOG-reactive IL-17-producing and IFN&#947;-producing CD4 cells. These results suggest that Mnk1/2 may play a minimal role in T cell development and function but may control non-T cell lineages to regulate TH1 and TH17 differentiation in vivo. </p><p>To determine the effect of constitutive mTOR complex 1 activity on anti-bacterial CD8 responses, we investigated mice with T cell-specific deletion of TSC1, a suppressor of mTOR complex 1 activity. Using an established model system of transgenic (OT1) CD8 cell adoptive transfer and challenge with Listeria monocytogenes expressing a cognate antigen, we found that TSC1 deficiency impairs antigen-specific CD8 responses. Fewer TSC1-deficient OT1 cells were present in the peripheral blood and spleen at the peak of the response and fewer memory cells were found at later time points, in individual and competitive adoptive transfer experiments with WT counterparts. Weak expansion of TSC1-deficient cells was correlated with defects in survival and proliferation in vivo, while exaggerated contraction was associated with an increased ratio of SLECs to MPECs in the effector cell population. This perturbation in effector-memory differentiation was concomitant with enhanced T-bet expression and decreased Eomes expression among activated TSC1 KO cells. Upon competitive adoptive transfer with WT counterparts and antigen re-challenge, TSC1-deficient memory cells showed moderate defects in expansion but not cytokine production. Taken together, these findings provide direct evidence of a CD8 cell-intrinsic role for TSC1 in regulating antigen-specific primary and memory responses.</p><p>In sum, findings from these studies provide deeper insight into the regulation of T cell function by DAG-mediated pathways, and may have implications for the design of immune-modulation strategies during vaccination, autoimmunity and cancer immunotherapy.</p> / Dissertation

Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5' cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4E(S209A)). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca2+ imaging experiments on dorsal root ganglion neurons, NGF-and IL-6-induced increases in excitability were attenuated in neurons from eIF4ES209A mice. These effects were recapitulated in Mnk1/2(-/-) mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4ES209A and Mnk1/2 (-/-) mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2-eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain.