Induction of apoptosis by PKCdelta: A tail of nuclear transport.

摘要

Our laboratory has previously demonstrated that PKCδ is required for mitochondrial-dependent apoptosis in salivary epithelial cells. To further understand the contribution of PKCδ to cell death, the present studies have characterized the subcellular distribution of PKCδ in apoptotic cells, investigated the role of nuclear localization and the mechanism by which PKCδ localizes to the nucleus, and uncovered a potential nuclear function of PKCδ during apoptosis. The first aim of this project was to determine the localization of PKCδ during etoposide induced apoptosis. We determined that nuclear translocation of PKCδ was observed as early as four hours after etoposide treatment and correlated temporally with nuclear accumulation of the PKCδ catalytic fragment (CFδ). The second aim of this project was to unravel the structural requirements for nuclear translocation during etoposide induced apoptosis. Transient transfection of the wild type or kinase deficient CFδ (CFδ or KNCFδ) resulted in their nuclear accumulation in the absence of an apoptotic stimulus. We have identified and mapped a functional nuclear localization signal (NLS) in the carboxyl-terminus of PKCδ that is required for nuclear import of both full-length and catalytic fragment PKCδ and is sufficient for driving nuclear import of a heterologous protein. The third aim of this project was to determine if nuclear localization of PKCδ is sufficient to induce apoptosis and required for etoposide induced apoptosis. Expression of CFδ, but not KNCFδ, is sufficient to induce apoptosis in salivary epithelial cells. Mutations within the NLS of CFδ inhibited nuclear accumulation and its ability to induce apoptosis demonstrating the requirement of nuclear localization for induction of apoptosis. Expression of KNCFδ protected cells from etoposide-induced apoptosis, but not when mutated in the NLS, indicating that nuclear PKCδ activity is also required for genotoxin-induced apoptosis. These results demonstrate that nuclear accumulation of the active CFδ is necessary and sufficient for the induction of apoptosis and suggests that nuclear PKCδ activity is required for induction of the mitochondria-dependent apoptotic pathway. Finally, the fourth aim of this project was to determine a functional role for PKCδ in the nucleus of apoptotic cells. PKCδ was found to associate with Stat1 which correlated temporally with nuclear serine 727 phosphorylation of Stat1 during apoptosis. Induction of apoptosis by PKCδ required both serine 727 and tyrosine 701 phosphorylation of Stat1 demonstrating an important role for the Stat1 signaling pathway downstream of PKCδ.