N-terminal V1 domain of PKCθ is essential for IL-2 transactivation in Jurkat T cells
While the N-terminus of conventional PKC isoenzymes contains the pseudosubstrate region, important for auto-inhibition, this region is rather variable in the subfamily of novel PKCs and has been implicated in isoenzyme-specific functions [10]. We addressed its relevance for PKCθ function by exchanging the sequence of exon 2, which encodes for the corresponding variable region (named V1). The sequence of the exon 2 replacing amino acids was chosen based on the PaptorX structure prediction program (http://raptorx.uchicago.edu). This E2mut extension, albeit being a longer amino acid sequence than the wild type exon 2 encoded sequence, revealed to be the best fit of a highly unstructured domain not affecting the overall structure of the adjacent C2-like domain encoded in exon 3 of PKCθ. Of note, this E2-replacement mutation does not target the conserved C2-like domain and the tyrosine (Y) 90 residue within (Fig. 1a), whose phosphorylation by Lck regulates membrane translocation of PKCθ and downstream transcription factor activation events [7, 18]. Thus, the Y90F mutation impairs the ability of the catalytically active PKCθ-A148E mutant to induce NFAT as well as NF-κB activation. Additionally, the other five phosphorylation sites (T219, T538, S676, S68, and S695) that have been reported to play critical roles in the regulation of PKCθ function and downstream signaling (reviewed in 9) are still present in their wild type form in the PKCθ-E2mut version (Fig. 1a). We tested the effect of the E2-replacement mutation in context of the constitutively active PKCθ mutant generated by the established Ala to Glu (A148E) exchange within the pseudosubstrate sequence to introduce a negative charge that mimics the presence of a phosphate at this location. This A/E background was used, since due to the high levels of endogenous PKC family member expression in Jurkat T cells, phorbol ester and ionomycin treatment to mimic TCR stimulation is not suitable to address the function of ectopically expressed PKCθ versions. Our experiments showed that, the E2-mutated PKCθ-A148E failed to induced IL-2 transactivation in transfected ionomycin-stimulated Jurkat T cells (Fig. 1b). The E2-mutation abolished also the capacity of the constitutively active PKCθ-A148E mutant to transactivate NFAT and AP-1 when assessed with a NFAT-AP1-dependent promoter luciferase reporter assay (Fig. 1c). However, due to endogenous PKCθ in Jurkat T cells, we could not convincingly show equal expression level of the ectopically PKCθ-A148E/E2wt and PKCθ-A148E/E2mut protein by immunoblot in transfected Jurkat T cells. Therefore, we decided to express a range of levels of PKCθ-A148E/E2wt and PKCθ-A148E/E2mut in Jurkat T cells and analyze the transfected cells in the NFAT-AP1-dependent promoter luciferase reporter assay. While we observed a dose-dependent increase of the luciferase signal when Jurkat T cells were transfected with PKCθ-A148E/E2wt, we did not detect signals above control transfected cells at any of the tested PKCθ-A148E/E2mut plasmid concentrations (Fig. 1c), suggesting that the “normal” N-terminus is essential for PKCθ function in T cells.
In addition, we analyzed the activity of PKCθ-E2wt and PKCθ-E2mut not harboring the A148E mutation in cells that lack endogenous PKCθ expression. Therefore, HEK-293T cells were co-transfected with the NFAT-AP1-dependent promoter luciferase reporter construct and either the E2wt or the E2mut version of PKCθ. Expression of PKCθ-E2mut impaired NFAT-AP1 transactivation in this cellular system, even though PKCθ-E2wt and PKCθ-E2mut protein expression was comparable when determined by immunoblot (Fig. 1d). Of note, however ionomycin/phorbol ester treatment seemed to activate endogenous kinases other than PKCθ since a strong activation of the NFAT-AP1-dependent promoter luciferase reporter construct was observed already in HEK-293T cells that were co-transfected with the empty control plasmid. Thus, ectopic expression of PKCθ-E2wt only marginally increased the transactivation. Nevertheless, ectopic expression of PKCθ-E2mut suppressed NFAT-AP1 transactivation, suggesting a dominant negative effect of the mutant on other PKCs. Altogether, in ectopic expression analysis in HEK-293T or Jurkat T cell lines, respectively, PKCθ-E2mut, when expressed at similar levels to the wt enzyme, shows a loss-of-function phenotype.
Mice bearing the PKCθ-E2 mutation show reduced PKCθ expression on protein level
After having observed the importance of PKCθ’s N-terminus for T cell activation events in cell lines using overexpression of a mutant version of PKCθ, we wanted to confirm this finding under more physiological conditions in vivo. Therefore, a mouse line carrying the mutated version of exon 2 (PKCθ-E2mut) was generated by targeted mutation in embryonic stem cells. PKCθ-E2mut mice were born following the expected Mendelian frequency with no obvious difference in viability and fertility, normal growth and weight development (unpublished observations). First, we analyzed the expression of the mutated version of PKCθ in T cells of the newly generated mouse line. At the mRNA level, quantitative real-time PCR did not reveal significant differences between the expression of wild-type (wt) and E2mut PKCθ in isolated CD4+ T cells, (Fig. 2a). However, at the protein level, there was reduced expression of the PKCθ-E2mut protein, which is 43 amino acids larger than its wild type counterpart (707aa; Fig. 2b). To address whether the E2 mutation impairs PKCθ protein stability, CD4+ T cells of wild type and PKCθ-E2mut mice were treated for different times with cycloheximide, which inhibits protein synthesis. PKCθ and as internal control actin protein levels were examined by western blot analysis. Protein levels of both PKCθ versions strongly decreased between 6 and 12 h of cycloheximide treatment (in relation to untreated cells). However, no significant differences in protein turnover rate of PKCθ-E2mut protein could be observed in these experiments (Fig. 2c).
PKCθ-E2 mutation impairs T cell development in vivo
Next, we addressed whether mice expressing PKCθ-E2mut instead of wt PKCθ show any abnormalities in their immune status, especially within the T cell compartment since it has been reported that positive selection during thymocyte development is affected in PKCθ-deficient mice [19, 20]. In line with these previous studies, we observed reduced frequencies of CD4 and CD8 single positive thymocytes in mice lacking PKCθ (Fig. 3a and Additional file 1: Figure S1), which was reflected by a decreased percentage of peripheral mature T cells (Fig. 3b). Even though CD3+ frequency among lymphocytes was lower in both genotypes it only reached statistical significance in case of PKCθ-E2mut mice. Of note, PKCθ-E2mut mice showed similar impairment in thymic development as PKCθ-deficient mice (Fig. 3a). Flow cytometric analyses of thymocytes showed that there was a strong and comparable reduction in Foxp3+CD25+CD4+ natural regulatory T cells (nTreg) in PKC-deficient and PKCθ-E2mut mice as compared to wt mice. Correspondingly, a reduction in Treg frequency was also observed in secondary lymphoid organs of both the analyzed PKCθ genotypes (Fig. 3b and Additional file 2: Figure S2), which has been already published for PKCθ-deficient mice [21, 22]. Nevertheless, currently it is not clear whether Treg-intrinsic PKCθ expression or its expression by conventional T cells is critical for thymic Treg development. Both sound reasonable, since induction of Foxp3 expression and Treg differentiation require increased signal strength [23], which might be reduced when PKCθ is absent. However, Treg development also depends on IL-2 provided by T cells in the thymus [24], whose expression is reduced when CD4+ T cells lack PKCθ. In contrast to the altered Treg compartment, however, naïve/memory distribution among CD4+ as well as CD8+ T cells in the periphery of PKCθ-deficient and PKCθ-E2mut mice was comparable to wt mice (Additional file 3: Figure S3). Furthermore, no gross changes in other immune cell compartments such as B cells, neutrophils and macrophages were detected (data not shown) as also no significant differences in overall spleen/lymph node cell counts (Fig. 3b).
Reduced activation of CD4+ T cells expressing PKCθ-E2mut
Mice deficient in PKCθ not only show a defect in thymic positive selection, as mentioned above, but also have a severe impairment in peripheral CD4+ T cell activation, especially regarding IL-2 expression [3, 4]. Moreover, PKCθ has been implicated in T cell survival, which was reduced when T cells lacked PKCθ expression [25, 26]. In contrast, we observed comparable survival of CD4+ T cells regardless of their genotype by Annexin V/7AAD staining and flow cytometry after overnight in culture - either non-stimulated or stimulated with anti-CD3 and anti-CD28 antibodies (Fig. 4a). But unlike in the published studies, we first cultured the cells only overnight and not for 36 or 72 h and second, used non-sorted splenocytes instead of isolated CD4+ T cells and identified CD4+ T cells thereafter by flow cytometry. Nevertheless, we observed reduced upregulation of the early activation markers CD25 and CD69 upon stimulation overnight when T cells expressed the mutant form of PKCθ or lacked PKCθ expression (Fig. 4b).
Moreover, IL-2 expression of PKCθ-deficient as well as PKCθ-E2mut CD4+ T cells was impaired at both the mRNA and protein level (Fig. 4c and Additional file 4: Figure S4). This finding is in agreement with publications reporting that exogenous IL-2, which acts as an extrinsic survival factor, did overcome the survival defects of PKCθ-deficient CD4+ T cells. Hence, we assume reduced viability secondary to lower IL-2 expression at later time points. Of note, the phenotype of PKCθ-E2mut CD4+ T cells did resemble the defects in early activation response and IL-2 expression observed for PKCθ-deficient CD4+ T cells (Fig. 4b, c), supporting the importance of the N-terminal V1 domain for PKCθ-mediated functions. However, we cannot rule out that this phenotype is caused by reduced PKCθ protein amount (as shown in Fig. 2b). Nevertheless, the results obtained with CD4+ cells of PKCθ-E2mut mice are in line with our findings with HEK-293T cells described above (Fig. 1d), where comparable expression of wt and E2-mutant PKCθ protein was detected.