Identification of the amino acid sequence motif for conventional PKC-mediated regulation of NKp46 surface expression

Abstract

A number of surface receptors expressed on NK cells are related to the regulation of NK cell activity and characterized by either inhibitory or activating properties. Natural cytotoxicity receptors (NCR) are one major family of activating receptors involved in NK cytotoxicity. The three family members of NCR are NKp46, NKp44, and NKp30. The surface density of these receptors might vary with the activation state of NK cells, and their density may directly correlate with their natural cytotoxicity. In this study, we investigated the regulation of NKp46 expression and determined the amino acid sequence motif for protein kinase C (PKC)-mediated regulation of its surface expression. We produced stable cell lines expressing full-length NKp46 and investigated the change in expression after PMA or IL-2 treatment using flow cytometry, RT-PCR, and immunoblotting methods. Expression of NKp46 on Jurkat T-cell transfectants appeared to be induced by PMA treatment until 8-h post-treatment and then gradually decreased afterwards to levels that were less than those measured at pretreatment. Parallel to surface expression of NKp46, total NKp46 protein expression also appeared to fluctuate after PMA treatment, but the expression of mRNA transcripts was not significantly affected. Experiments with mutant NKp46-expressing stable cell lines demonstrated that Ser288 might be critical for the surface expression of NKp46 and the PKC-mediated regulation of NKp46 expression. However, NKp46 surface expression was not influenced by IL-2 in stable cell lines expressing wild-type and mutant NKp46