STRUCTURE AND FUNCTION ANALYSIS OF PAR-4

Par-4 is a leucine zipper domain protein that induces apoptosis on its own in certain cancer cells and in Ras-transformed cells, but not in normal or immortalized cells. Par-4 induces apoptosis by activation of the Fas death receptor pathway and co-parallel inhibition of NF-B transcription activity. Cells that are resistant to apoptosis by Par-4 alone, however, are greatly sensitized by Par-4 to the action of other pro-apoptotic insults such as growth factor withdrawal, TNF, ionizing radiation, intracellular calcium elevation, or those involved in neuronal degeneration such as Alzheimer\u27s, Parkinson\u27s, Huntington\u27s and Stroke. Previous studies have suggested that the apoptosis-sensitization potential of Par-4 is dependent upon inhibition of PKC or WT1 cell survival function by direct interaction between the leucine zipper domain at the carboxy-terminus of Par-4 and the zinc finger domains of PKC or WT1. In this study, I performed structure-function analysis using GFP-fusion proteins and deletion mutants to identify the functional localization and domains of Par-4 that are essential for apoptosis induction. My findings suggest that apoptosis by Par-4 is dependent on its translocation to the nucleus for induction of apoptosis. A bipartite nuclear localization signal sequence corresponding to amino acids 137-155 was necessary for nuclear translocation of Par-4. Importantly, the core residues 137-204 in the center part of Par-4 were necessary and sufficient to induce Fas pathway activation, inhibition of nuclear NF-B transcription activity and apoptosis. These findings imply that binding of Par-4 via its leucine zipper domain to other proteins is dispensable for apoptosis by Par-4

Identification of a Unique Core Domain of Par-4 Sufficient for Selective Apoptosis Induction in Cancer Cells

Recent studies indicated that the leucine zipper domain protein Par-4 induces apoptosis in certain cancer cells by activation of the Fas prodeath pathway and coparallel inhibition of NF-κB transcriptional activity. However, the intracellular localization or functional domains of Par-4 involved in apoptosis remained unknown. In the present study, structure-function analysis indicated that inhibition of NF-κB activity and apoptosis is dependent on Par-4 translocation to the nucleus via a bipartite nuclear localization sequence, NLS2. Cancer cells that were resistant to Par-4-induced apoptosis retained Par-4 in the cytoplasm. Interestingly, a 59-amino-acid core that included NLS2 but not the C-terminal leucine zipper domain was necessary and sufficient to induce Fas pathway activation, inhibition of NF-κB activity, and apoptosis. Most important, this core domain had an expanded target range for induction of apoptosis, extending to previously resistant cancer cells but not to normal cells. These findings have identified a unique death-inducing domain selective for apoptosis induction in cancer cells (SAC domain) which holds promise for identifying key differences between cancer and normal cells and for molecular therapy of cancer