The goal of the proposed research is to determine the structural and biochemical factors involved in converting cytosolic human K-ras 4B protein into membrane-bound protein. Oncogenic forms of human K- ras 4B protein are frequently detected in human cancers, and it is believed that oncogenic K-ras 4B protein may play a crucial role in the pathogenesis of these malignancies. Since oncogenic K-ras 4B protein must become membrane associated in order to malignantly transform cells, elucidation of the factors which enable K-ras 4B protein to become membrane associated may improve our understanding of the mechanism of action of ras, and may ultimately allow us to develop rational therapeutic interventions that might inhibit K-ras 4B protein membrane association, and prevent it from contributing to human carcinogenesis. Ras proteins are synthesized in the cytosol as inactive precursors and must become membrane associated to cause malignant transformation. The factors which enable ras proteins to become membrane associated are unknown. However, recent studies suggest that certain post-translational modifications (PTMS) of ras proteins may promote or stabilize membrane association. In the case of H-ras, and N-ras proteins, these PTMS include palmitylation, isoprenylation, carboxyl methylation and truncation; but, the relative importance of each of these modifications to H- and N-ras protein membrane association and biological activity is unknown. Less is known about the PTMS of K-ras 4B protein. However, several major differences between K-ras 4B and the other ras proteins suggest that the factors which enable K-ras 4B to become membrane associated and biologically active, may significantly differ from the factors believed to be important for H- or N-ras proteins. First, unlike H- or N-ras, K-ras 4B is not palmitylated. Second, K-ras 4B (but not H- or N-ras) contains a large number of positively charged residues (including a stretch of 6 contiguous lysines) in its C-terminal region. Third, K-ras 4B is converted from cytosolic to membrane-bound protein much more rapidly than H-ras protein. Fourth, K-ras 4B (but not H- or N-ras) is phosphorylated in vivo by a cAMP-dependent protein kinase (similar phosphorylation of the ras-related protein, rap la, affects rap la membrane association). Since oncogenic K-ras 4B protein is the most frequent oncogene product detected in human cancers, studies aimed at delineating the factors which enable this protein to become membrane associated and biologically active are crucial.
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