Functional comparison of protein domains within aPKCs involved in nucleocytoplasmic shuttling

The atypical protein kinases C (PKC) isoforms iota and zeta play crucial roles in regulation of signaling pathways related to proliferation, differentiation and cell survival. Over the years several interaction partners and phosphorylation targets have been identified. However, little is known about the regulation of atypical aPKC isoforms. To address this question, we performed a comparative analysis of atypical aPKC iota/lambda and zeta in MDCK cells. By using green fluorescence protein (GFP) fusion proteins containing the full-length or truncated proteins, we were able to recognize differences in subcellular localization and nucleocytoplasmic shuttling of both isoforms. We show, that an earlier described nuclear localization sequence (NLS), plays a role in the regulation of atypical aPKC zeta but not in aPKC iota, despite the fact that it is present in both isoforms. Leptomycin B treatment induces accumulation of GFP-fusion protein of both isoforms in the nucleus. Regardless, the loss of the NLS only decreases shuttling of aPKC zeta, while aPKC iota remains unaffected. In addition, we identified the hinge region as a potential regulator of localization of atypical PKCs. With a set of chimeric proteins we show that the hinge region of aPKC iota mediates nuclear localization. In contrast, the hinge region of aPKC zeta causes exclusion from the nucleus, indicating two different mechanisms leading to isoform specific regulation. Taken together, we show for the first time, that the atypical isoforms aPKC iota and zeta underly different mechanisms regarding their regulation of subcellular localization and translocation into the nucleus in MDCK cells. (C) 2012. Published by The Company of Biologists Ltd.