The ras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions such as Gly 12 and Gln 61 and is a major cause of human tumors and malignant transformation of normal cells and maturation of oocytes. Oncogenic and activated normal p21 utilize divergent pathways. P21 interacts with the raf protein resulting in activation of the MEK protein and MAP kinase (MAPK) in a mitogenic signaling pathway. It activates a newly-identified independent signaling pathway by interacting with jun kinase (JNK) and jun. Using computer-based molecular modeling, that has identified effector domains of p21 it is proposed to identify the regions of the ras-binding domain of raf that are effector domains, to synthesize peptides corresponding to these domains and to test these peptides for their abilities to inhibit oncogenic and insulin-activated normal protein-induction of oocyte maturation. To probe differences between the oncogenic and normal mitogenic pathways, correlation will be followed between activation of JNK and jun and oocyte maturation induced by microinjected transforming p21 or by insulin-induced activated normal p21 to determined if transforming p21 preferentially activates the JNK pathway. To explore the relationship between the raf/MAPK and JNK pathways, dominant negative mutants of MEK and MAPK will be co-injected into oocytes with JNK to determine if they inhibit JNK-stimulated maturation. Also, A JNK peptide inhibitor will be co-injected with the transforming p21 to determine if it blocks activation of MAPK. Since oncogenic but not normal p21 requires protein kinase C (PKC), the effect of a PKC inhibitor on JNK-induced oocyte maturation will be determined as will be the effect of dominant negative MEK and MAPK mutants on PKC-induced oocyte maturation. Since some p21 peptides have been found to inhibit transforming -21- but not insulin-activated normal p21-induced oocyte maturation, the sites of inhibition of these peptides will be identified by correlating their IC50 values for oocyte inhibition with those for their inhibition of the binding of p21 to bead- bound JNK, jun and raf. Also, bead-bound peptides will be incubated with cell lysates of ras-transformed cells to detect proteins with which they interact.
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