pp32 is a lineage-independent nuclear phosphoprotein expressed at high levels in stem-like self-renewing cell populations and neoplastic cells, but not in terminally differentiated cells. Expression of pp32 appears to increase with malignant potential in tumors such as prostate cancer. pp32 has a leucine zipper and an acidic domain, suggesting interaction with other nuclear elements. Previous work under this grant established three functional activities for pp32 which may relate to its role in self-renewing and neoplastic cells: pp32 suppresses transformation of fibroblasts cotransformed by ras with myc, jun, mutant p53, or E1a; pp32 modulates cellular sensitivity to drug- induced programmed cell death; and pp32 may modulate nuclear shape and size. Studies proposed for the next funding period seek to understand structural- functional relationships in pp32 and relate these to its increased expression in neoplasia. The hypotheses to be tested are: (1) that pp32 functions are associated with discrete elements with pp32 sequence; (2) that one or more functionally significant pp32 sequence elements or motifs will govern the interaction of pp32 with one or more additional protein molecules; and (3) that interaction of pp32 with one or more additional protein molecules is obligatory for pp32 to confer resistance to transformation or apoptosis, to modulate nuclear morphology, or potentially modulate transcriptional activity. The detailed aims are:
(Aim one) to determine structure-function relationships in pp32 by mutational analysis through truncation, deletion, and point mutation with subsequent testing for the ability to suppress transformation, protect against programmed cell death, and modulate nuclear size;
(Aim two) to identify and characterize unknown molecules which bind to pp32 and to test the ability of known molecules such as fos, jun myc, e1a and p53 using a two-hybrid system, immunoprecipitation, and, for unknown molecules only, affinity chromatography;
and (Aim three) to characterize the regulation of pp32 expression in normal and neoplastic cells in primary cultures of normal and neoplastic cells, normal cell lines, and neoplastic cell lines studied to determine the critical factors which regulate pp32 levels examining transcriptional activation, mRNA half-lives, protein synthetic rates, and protein half-lives.
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