Expression and localization of atypical PKC isoforms in liver parenchymal cells

Abstract

AbstractMembers of all three classes of the protein kinase C (PKC) family including atypical PKCzeta (PKCζ) are involved in central functions of liver parenchymal cells. However, expression and localization of PKCiota (PKCι), the highly homologous atypical PKC (aPKC) isoform, in hepatocytes is unknown to date. PKCζ and PKCι were cloned from human and rat liver and fused to fluorescent protein tags (YFP). The sequence of full-length rat PKCι is not yet known and was cloned from cDNA of hepatocytes by the use of degenerated primers. PKCζ-YFP and PKCι-YFP (human and rat) were expressed in HeLa or HEK293 cells and used to test the specificity of seven aPKC antibodies. Two antibodies were PKCι-specific and two were specific for PKCζ in immunofluorescence and Western blot analysis. Subcellular localization was analyzed by immunofluorescence in isolated rat and human hepatocytes and liver sections. Low immunoreactivity for aPKCs was found at the sinusoidal membrane and in the cytosol. The highest density of PKCι as well as PKCζ was found at the canalicular membrane in co-localization with ABC-transporters, such as bile salt export pump or multidrug resistance-associated protein 2. This topology suggests a specific function of aPKCs at the canalicular membrane in addition to their known role in cell polarity of epithelial cells.