Amino-acid sequence motifs for PKC-mediated membrane trafficking of the inhibitory killer Ig-like receptor

Abstract

Activation-induced upregulation of inhibitory killer Ig-like receptor (KIR) is regulated by protein kinase Cs (PKCs). Conventional PKCs increase KIR expression on the post-transcriptional level by increasing the recycling of surface molecules and endoplasmic reticulum (ER)-Golgi processing. PKCdelta plays a role in the secretion of cytoplasmic KIR through lytic granules. In this study, we identified amino acid sequence motifs associated with PKC-mediated KIR membrane trafficking by systematic mutagenesis. Mutations of Y(398) and HLWC(364) completely inhibited the PMA-induced increase of KIR molecules at surface as well as total protein levels, indicating that these are associated with ER-Golgi processing and sorting to plasma membrane through lytic granules. Mutations of Y-based motif, including Y(398), acidic region (PE(394)), dileucine motif-like region (IL(423)) and PKC-phosphorylatable S(415) caused a blockade of surface KIR endocytosis after PKC stimulation. Mutation of T(145) caused an accumulation of mutant proteins in late endosomes and lysosomes after PKC activation, suggesting that T(145) might be related to the recovery of endocytosed KIR to the surface membrane. We also demonstrated that PKCs could directly phosphorylate the KIR cytoplasmic tail by means of western blot and in vitro kinase assay, implying that phosphorylation status of KIR cytoplasmic tail can direct the fate of surface KIR molecules. Taken together, various sequence motifs are implicated in the PKC-mediated post-transcriptional upregulation of KIR, and each of these motifs work in different steps after PKC activation.