The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.
IntroductionDendritic cells (DCs) are the most potent antigen-presenting cells (APCs) of the immune system and are uniquely capable of stimulating clonal expansion of naive T cells, providing a plethora of costimulating molecules and cytokines. 1,2 However, there is also experimental evidence for a function of DCs in the induction of tolerance. 3,4 Defining the mechanisms responsible for this phenomenon has become one of the most intriguing challenges in immunology. It is widely assumed that various subpopulations of DCs are involved in the initiation and maintenance of peripheral tolerance. In addition, the generation of regulatory T cells (Tregs) induced by DCs plays a critical role for maintenance of peripheral tolerance. Currently, various T-cell populations are considered to be regulatory T cells, including CD4 ϩ CD25 ϩ Tregs T R 1 and T H 3 cells. 5-9 These heterogeneous cell populations are capable of suppressing effector T-cell functions through diverse contact-dependent and/or -independent mechanisms, specific for the respective regulatory cell type. [5][6][7][8][9] Previously, we have demonstrated that IL-10-modulated tolerogenic DCs (IL-10DCs) induce anergic CD4 ϩ and CD8 ϩ T cells with antigen-specific regulatory properties (iTregs) inhibiting activated CD8 ϩ and CD4 ϩ effector T cells. [10][11][12] These iTregs are characterized by an G 1 arrest of the cell cycle mediated via high expression of the cyclin-dependent kinase (cdk) inhibitor p27 Kip1 . 13 Addition of IL-2 and blocking of the CTLA-4 pathway abolished the state of anergy and also resulted in a loss of suppressive properties of iTregs, indicating an interaction between cell-cycle regulation, mechanisms of signal transduction, and suppressor function of iTregs induced by tolerogenic IL-10DCs. 13 Mitogen-activated protein kinases (MAPKs) play crucial roles in mediating cellular responses to various extracellular signals. Currently, 4 major subgroups of the MAPK family have been identified: stress-activated protein kinases (SAPKs)/Jun N-terminal kinases (JNK1/2), p38 kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and, more recently, ERK5. [13][14][15] It was demonstrated that activation of the MAPKs ERK 16 and JNK 17 is reduced in anergic T cells induced in the absence of costimulation due to a blocked activity of p21 RAS in the case of ERK. Some reports described a reduced activity of p38 in anergy but so far the function of these signal transduction events in Tregs has not been defined.In this study, we show that the activity of the MAPK p38 and its downstream effectors MAPK-activated protein kinases 2/3 (MAP-KAP-K2/3) is markedly enhanced in iTregs induced by IL-10DCs, whereas the activation of the MAPKs JNK and ERK is significantly reduced. Inhibition of p38 in iTregs induces a markedly reduced expression of the cdk inhibitor p27 Kip1 , resulting in cell-cycle progression and complete loss of regulatory function. Taken together, these data suggest a critical role of altered MAPK signaling in iTregs, affecting the cross...
We have identified protein kinase C-c (PKC-6) as a novel suppressor of neoplastic transformation caused by the v-raf oncogene. PKC-Y overexpression drastically retards proliferation, abolishes anchorage-independent growth, and reverts the morphological transformation of v-raf-transformed NIH-3T3 cells. The molecular basis for this effect appears to be a specific induction of iunB and egr-1 expression, triggered synergistically by PKC-5 via a RafIMeklMAPK-independent mechanism and v-raf. junB-promoterlCAT assays revealed that PKC-c directly targets the junB promoter. The induction of iunB and egr-1 is linked to the v-raf transformation-suppressing effect of PKC-c as constitutive expression of iunB and egr-1 but not of c-jun also abolishes anchorage-independent growth of v-raf-transformed NIH-3T3 cells. Moreover, junB overexpression leads to a retardation of proliferation in these cells. PKC-5 interferes with the serum inducibility of an AP-1 reporter plasmid in v-raf-transformed NIH-3T3 cells, indicating that PKC-5 antagonizes transformation and proliferation by down-modulating AP-1 function via induction of junB. In summary, our data suggest that PKC-Y counteracts v-raf transformation by modulating the expression of the transcription factors junB and egr-1.
Dendritic cells (DCs) are the most potent APCs of the immune system. Understanding the intercellular and intracellular signaling processes that lead to DC maturation is critical for determining how these cells initiate T cell-mediated immune processes. NO synthesized by the inducible NO synthase (iNOS) is important for the function of murine DCs. In our study, we investigated the regulation of the arginine/NO-system in human monocyte-derived DCs. Maturation of DCs induced by inflammatory cytokines (IL-1β, TNF, IL-6, and PGE2) resulted in a pronounced expression of neuronal NOS (nNOS) but only minimal levels of iNOS and endothelial NOS were detected in human mature DCs. In addition, reporter cell assays revealed the production of NO by mature DCs. Specific inhibitors of NOS (N-nitro-l-arginine methyl ester) or of the NO target guanylyl cyclase (H-(1,2,4)-oxadiazolo [4,3-a] quinoxalin-1-one) prevented DC maturation (shown by decreased expression of MHC class II, costimulatory and CD83 molecules and reduced IL-12 production) and preserved an immature phenotype, indicating an autocrine effect of nNOS-derived NO on human DC maturation. Notably, inhibitor-treated DCs were incapable of inducing efficient T cell responses after primary culture and generated an anergic T cell phenotype. In conclusion, our results suggest that, in the human system, nNOS-, but not iNOS-derived NO, plays an important regulatory role for the maturation of DCs and, thus, the induction of pronounced T cell responses.
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