The ras-oncogene-encoded p21 protein, a G-protein, that is normally activated by binding GTP in place of GDP, is present in all eukaryotic cells and is known to be critical in the regulation of the cell cycle becomes oncogenic when amino acids are substituted at critical positions such as Val-for-Gly 12 and Leu-for-Gin 61. The oncogenic but not the normal, protein, causes cell transformation when microinjected into NIH- 3T3 cells and maturation of Xenopus laevis oocytes. To determine the specific activating conformational changes, conformational analysis, using a combination of molecular dynamics based on the computer program DISCOVER and an electric dipole moment-Monte Carol method (EDMC) based on the computer program ECEPP (Empirical Conformational Energies of Peptides Program), will be performed on the normal p21-GDP complex, the activated p21-GTP complex, and a series of non-oncogenic and oncogenic mutant proteins. Comparison of the average structures for the non- oncogenic and oncogenic proteins will highlight regions of major structural differences between them. Peptides from these regions will be synthesized and tested for their abilities to block oncogenic p21 protein-induced oocyte maturation. Thus, far, six different p21 segments have been so identified, and peptides from three of these regions (35-47, 96-110, and 115-126) have been found to block oncogenic p21-induced oocyte maturation. To identify the protein targets of p21 and these peptides intracellularly, photoaffinity-labeled Val 12-p21 photoaffinity- labeled active peptides will be introduced into cells to enable them to label their targets covalently. These proteins will be identified by immunoprecipitation, SDS-PAGE, and microsequencing. A protein of MW 43 kda has been isolated, using this technique, that may be MAP-2 kinase or MAP kinase kinase (MKK) and will be microsequenced and blotted with antibodies to these proteins. The inhibitory peptides and other anti-ras agents completely block oncogenic p21-induced oocyte maturation but not maturation induced by insulin which activates normal cellular ras. Also, protein kinase C has been found to be an essential target of oncogenic but not normal p21. These findings suggest that a major divergence in signal pathways occurs between oncogenic and activated normal ras- promoted p34-cyclin activation of this enzyme, inhibitors of the cyclin system will be tested to determine if they selectivity block insulin but not Val 12-p21-induced oocyte maturation.
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