Protein kinase C (PKC)θ is an established component of the immunological synapse and has been implicated in the control of AP-1 and NF-κB. To study the physiological function of PKCθ, we used gene targeting to generate a PKCθ null allele in mice. Consistently, interleukin 2 production and T cell proliferative responses were strongly reduced in PKCθ-deficient T cells. Surprisingly, however, we demonstrate that after CD3/CD28 engagement, deficiency of PKCθ primarily abrogates NFAT transactivation. In contrast, NF-κB activation was only partially reduced. This NFAT transactivation defect appears to be secondary to reduced inositol 1,4,5-trisphosphate generation and intracellular Ca2+ mobilization. Our finding suggests that PKCθ plays a critical and nonredundant role in T cell receptor–induced NFAT activation.
Using model tumor T cell lines, protein kinase C (PKC) α has been implicated in IL-2 cytokine promoter activation in response to Ag receptor stimulation. In this study, for the first time, PKCα null mutant mice are analyzed and display normal T and B lymphocyte development. Peripheral CD3+ PKCα-deficient T cells show unimpaired activation-induced IL-2 cytokine secretion, surface expression of CD25, CD44, and CD69, as well as transactivation of the critical transcription factors NF-AT, NF-κB, AP-1, and STAT5 in vitro. Nevertheless, CD3/CD28 Ab- and MHC alloantigen-induced T cell proliferation and IFN-γ production are severely impaired in PKCα−/− CD3+ T cells. Consistently, PKCα-deficient CD3+ T cells from OVA-immunized PKCα-deficient mice exhibit markedly reduced recall proliferation to OVA in in vitro cultures. In vivo, PKCα-deficient mice give diminished OVA-specific IgG2a and IgG2b responses following OVA immunization experiments. In contrast, OVA-specific IgM and IgG1 responses and splenic PKCα−/− B cell proliferation are unimpaired. Our genetic data, thus, define PKCα as the physiological and nonredundant PKC isotype in signaling pathways that are necessary for T cell-dependent IFN-γ production and IgG2a/2b Ab responses.
We here investigate the crosstalk of PKC and PKA signaling during primary CD3 ؉ T-lymphocyte activation using pharmacologic inhibitors and activators in combination with our established panel of PKC isotype-deficient mouse T cells in vitro. PKC and PKA inversely affect the CD3/ CD28-induced IL-2 expression, whereas other PKC isotypes are dispensable in this signaling pathway. Gene ablation of PKC selectively results in a profound reduction of IL-2 production; however, complete abrogation of IL-2 production in these PKC ؊/؊ T cells was achieved only by simultaneous coactivation of the cAMP/PKA pathway in CD3 ؉ T cells. Conversely, the reduced IL-2 production in PKC inhibitor-treated T cells can be rescued by inhibition of the cAMP/PKA pathway in wild-type but not in PKC ؊ IntroductionFollowing T-cell receptor (TCR) stimulation, lymphocyte activation threshold is coordinated by a complex interplay of distinct signal transduction pathways. The "fine-tuning" of signaling cascades is considered as a crucial step during T-cell activation in order to result in adequate amplitude of an immune response. The molecular processes that regulate the precise level of the immune activation and their complex interpathway integration are, however, still poorly understood. Among several signaling pathways, protein kinase C (PKC) plays an important role in the cellular activation pathway downstream of the antigen receptor. PKC, in particular, is known to translocate to the immunologic synapse. 1 Once activated, PKC plays a critical role in TCR signaling. 2 This is confirmed by analysis of CD3 ϩ T cells of mice lacking PKC. Ex vivo, PKC-deficient T cells are mostly TCR unresponsive and deficient in the production of interleukin 2 (IL-2) due to an impaired transactivation of NF-B, AP-1, and NF-AT. [3][4][5] In strict contrast, the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway represents a negative regulatory mechanism that controls IL-2 expression in T cells. cAMP itself has been demonstrated to activate directly a class of cyclic nucleotide ion channels 6,7 as well as the Rap1 guanine exchange factors Epac1 and Epac2. 8 The principle cAMP receptor in lymphoid cells, however, is PKA. 9 cAMP is produced after TCR stimulation in T lymphocytes, suggesting the cAMP-mediated repression of T-cell activation may represent a physiologic negative feedback control mechanism. 10 Suppression of cAMP signaling is thus required for T-cell activation. 11 Among other mechanisms, cAMP inhibition is mediated by de novo transcriptional induction of phosphodiesterases (PDEs) upon T-cell activation. In T cells, PDE4 seems to play a particularly important role. 12,13 Increased PDE4 expression in T cells is associated with increased IL-2 production via its hydrolysis of cAMP. 12 Inversely, PDE4 blockade is shown to augment cAMP levels and thus known to abrogate cytokine secretion and proliferation in T cells through inhibition of NF-B and NF-AT and activation of CREB. 13 Several direct targets of cAMP/PKA signaling have be...
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