The ras oncogene-encoded p21 protein, vital in control of the cell cycle, becomes oncogenic when single amino acid substitutions (like Val-for-Gly 12) occur at critical positions in the polypeptide chain. Oncogenic, unlike normal, p21 causes cell transformation, maturation (meiosis) of Xenopus laevis oocytes and many human malignancies, making it important to devise ways to block its effects. Computer-based molecular modeling studies suggest that oncogenic amino acid substitutions cause changes in the conformation of domains of the protein. Synthetic peptides corresponding to three of these domains (35-47, 96-110 and 115-126) block oncogenic p21- but not insulin-activated normal p21-induced oocyte maturation. Val l2-p2l has unique direct interactions with jun and its kinase jun kinase (JNK), blocked by the 96-110 and 115-126 peptides. The JNK-jun pathway interacts strongly with the raf-MEK-MAP kinase (MAPK) pathway; dominant negative raf blocks JNK-induced maturation and a newly discovered JNK inhibitor, glutathione-S-transferase (GST), blocks raf-induced maturation. Since the x-ray structures of p21 bound to the ras binding domain of raf (RBD), SOS (guanine nucleotide exchange protein) and GAP (GTPase activating protein), have been determined, we will use molecular modeling studies to determine domains of these proteins that change conformation when bound to oncogenic p21, synthesize peptides corresponding to these domains and test their abilities to block Val 12-p2l selectively. All peptides found to block Val l2-p21 selectively in oocytes will be introduced into mammalian cancer cells either attached to leader sequences or encoded in plasmids to test their abilities to block cell proliferation. Similarly identified GST domains will be tested for their abilities to affect GST-JNK interactions. Since the Val 12-p2 1-induced raf and iNK pathways are closely linked, we will determine which proteins on each pathway cross-activate one another using protein-specific inhibitors that will also be assayed for their abilities to block the binding of Val 12-p21 to both raf and JNK.
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