The p53 tumor suppressor is the most frequent genetic lesion implicated in human cancers. Tumor growth in the presence of wild-type p53 may be caused by the loss of other tumor suppressor genes, the overexpression of p53 inhibitors, or the lack of p53 stimulators. Other possible causes may include factors that regulate p53 inhibitors and stimulators. Recently, an apoptotic stimulator protein of p53 (ASPP2) was shown to bind p53 and stimulate p53-dependent apoptosis and was missing in 9 out of 40 human breast carcinomas. An ASPP family member, iASPP, acts as an oncoprotein that inhibits p53 activity. If the expression of ASPP family members can regulate p53 activity, then the regulation of ASPP2 activity may influence p53-dependent apoptosis during tumor suppression. The C-terminus of ASPP2 was shown to interact with other proteins that modulate its role in regulating apoptosis and p53 activity. One of these interactions seems to be negatively regulated through the phosphorylation of tyrosine residues in the SH3 domain of ASPP2. Although these ASPP2-interacting proteins (ASbp) were identified using the yeast two-hybrid assay, fragments of the ASbps, not ASPP2, were used as bait to screen the cDNA libraries. Because of the diversity of ASbps and the modification of ASPP2's function, these data suggest that other ASbps may also bind and modulate ASPP's activity. These studies proposed will seek to answer the following questions: What other proteins are interacting with ASPP2 and how does this interaction influence its apoptotic function? The proposars hypothesis is that ASPP2 interacts with proteins, not identified, that modulate its stimulatory role for p53-dependent apoptosis. The following specific aims will investigate this hypothesis: 1) Isolate and identify genes that encode proteins that bind ASPP2 in the yeast two-hybrid system; 2) Confirm binding specificity between ASPP2 and putative ASbps in vivo using mammalian cell lines; 3) Determine the effects of ASPP2 stimulation of p53's gene transactivation and apoptotic induction. Consequently, by identifying new ASbps, this study will expand the current knowledge of ASPP2's p53-stimulatory function, identify mechanisms of cancer not due to mutant p53, and possibly elucidate other ASPP2 regulated pathways.
