Abstract
BackgroundProtein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line.MethodsThe present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-κB transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry.ResultsDespite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKCθ for a transactivation of NF-κB and NFAT transcription factors and, subsequently, robust IL-2 and IFN-γ expression.ConclusionThis phenotype is reminiscent of the PKCθ knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKCθ function in primary mouse T cells.
Highlights
Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and Nuclear factor of activation in T cells (NFAT)
Flow cytometric analysis of thymocyte populations in wild-type control and PKCθT219A knockin mice revealed no differences in the distribution of CD3, CD4/CD8 double-positive and CD4, CD8 single-positive cells, whereas PKCθ knockout mice showed reduced frequencies of CD4 and CD8 single positive thymocytes (Fig. 2a & Additional file 1: Figure S1), which is in line with previous studies
Since it has been reported that PKCθ deficiency affects the positive selection process in thymocyte development, leading to a lower thymic frequency of CD4 and CD8 single positive cells [12, 13, 18], we carefully checked if there are any abnormalities within the T cell compartment of PKCθT219A mice: our results clearly show no differences in T cell subset numbers and frequencies in thymus and periphery between wild-type control and knockin mice
Summary
PKC isotypes are activated by antigen receptors, costimulatory receptors such as CD28, cytokines and integrins, and their function is regulated by activation of upstream kinases and/or by subcellular localization, which depends on kinase:lipid and kinase: protein interactions, enabling them to phosphorylate specific protein substrates [1, 2]. PKCθ has been shown to translocate to the cell-cell contact site, the so-called immunological synapse (IS), after interaction of a T cell with an antigen-presenting cell (APC) [2]. Both the PI3-K/Vav and ZAP-70/SLP-76 pathways have been
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