Abstract
In T cells PKCθ mediates the activation of critical signals downstream of TCR/CD28 stimulation. We investigated the molecular mechanisms by which PKCθ regulates NFκB transactivation by examining PKCθ/β single and double knockout mice and observed a redundant involvement of PKCθ and PKCβ in this signaling pathway. Mechanistically, we define a PKCθ-CYLD protein complex and an interaction between the positive PKCθ/β and the negative CYLD signaling pathways that both converge at the level of TAK1/IKK/I-κBα/NFκB and NFAT transactivation. In Jurkat leukemic T cells, CYLD is endoproteolytically processed in the initial minutes of stimulation by the paracaspase MALT1 in a PKC-dependent fashion, which is required for robust IL-2 transcription. However, in primary T cells, CYLD processing occurs with different kinetics and an altered dependence on PKC. The formation of a direct PKCθ/CYLD complex appears to regulate the short-term spatial distribution of CYLD, subsequently affecting NFκB and NFAT repressional activity of CYLD prior to its MALT1-dependent inactivation. Taken together, our study establishes CYLD as a new and critical PKCθ interactor in T cells and reveals that antagonistic PKCθ/β-CYLD crosstalk is crucial for the adjustment of immune thresholds in primary mouse CD3+ T cells.
Highlights
The central role of PKCh in signal transduction pathways during an adaptive immune response has extensively focused on the exact biochemical mechanisms of PKCh function
The tumor suppressor gene CYLD encodes an evolutionary conserved and ubiquitously expressed protein of approximately 120 kDa and was originally discovered as gene mutated in familial cylindromatosis, an autosomal dominant inherited disease characterized by the development of multiple benign skin tumors, principally on the head and neck [11]
Association of CYLD with PKCh Considering the reciprocal phenotypes of Cyld- and PKCh/bdeficient T cells involving NFAT and NFkB transactivation we investigated a potential direct interaction between this enzymes
Summary
The central role of PKCh in signal transduction pathways during an adaptive immune response has extensively focused on the exact biochemical mechanisms of PKCh function (reviewed in [1,2,3]). The tumor suppressor gene CYLD encodes an evolutionary conserved and ubiquitously expressed protein of approximately 120 kDa and was originally discovered as gene mutated in familial cylindromatosis, an autosomal dominant inherited disease characterized by the development of multiple benign skin tumors, principally on the head and neck [11] It is a deubiquitylating enzyme (DUB) which removes mainly K63-linked polyubiquitin chains from several specific substrates, influencing in a negative way the activation status and/or spatial distribution of these target proteins in different signaling pathways. Numerous studies both in vitro and in vivo provided us with new insights in its established function as an important negative regulator of inflammatory responses, by counteracting the aberrant activation of NFkB signaling: Cyld2/2 animals spontaneously develop intestinal inflammation and autoimmune symptoms due to the constitutive activation of the TAK1/IKK/IkBa axis [12,13]; the study of Lim et al described a CYLD dependent negative NFkB regulation during bacteria induced lung inflammation in mice via deubiquitylation of TRAF6 and TRAF7 [14]; the same scientific group showed that Cyld knockout mice are protected from Streptococcus pneumonia infection and lethality via a negative crosstalk with p38 MAPK [15]; a synergistic crosstalk between the E3 ligase Itch and CYLD for TAK1 inactivation and termination of tumor necrose factor dependent inflammatory signaling was recently described [16]
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