Ras proteins serve as critical control elements in the response of cells to external signals. The stimulation of some plasma membrane receptors can lead to activation of Ras and a mitogenic response. Mutant, constitutively active, forms of Ras can lead to a persistent mitotic signal that is independent of receptor stimulation. Such Ras mutations are among the most common causative events in human tumors. In some specialized mammalian cell types, however, Ras activation leads to cell cycle arrest and differentiation. This suggests that cell type differences provide a context for the interpretation of Ras signals which may lead to either cell cycle progression or arrest and differentiation. One downstream pathway directly connecting Ras activation to changes in gene expression that are required for cell division has been uncovered. The key first step is activation of the protein kinase Raf1. This is initiated by a physical interaction between Raf1 and Ras. There are however, other Ras-mediated events that appear to be required for a full transformation or differentiation response, suggesting the involvement of additional Ras interaction partners. They have isolated and are continuing to study Rin1, a human protein that binds specifically to activated Ras and can compete with Raf1. There are additional parallels between Rin 1 and Raf1 behavior, including their interactions with 14-3-3 proteins. Tissue type expression of Rin1 is regulated over a wide range. In addition, some splice variant forms of Rin1 have altered properties. Their findings are consistent with Rin1 functioning as a regulated Ras effector, as a Raf1 competitor or as both. Interestingly, the rin 1 gene has been mapped to 11q13.2, a chromosomal position that is frequently amplified in squamous cell carcinomas and breast tumors. This proposal includes an analysis of Rin1 binding properties and function. The focus is on interactions that occur in mammalian cells (co-immunoprecipitations and co-immunofluorescence) and on the biological consequences of those interactions (effects on Ras-mediated transformation and differentiation). They will take advantage of both artificial and naturally occurring variants of Rin1 that have different binding properties. These studies, directed at the role of Rin1 function in Ras-mediated signal transduction, should facilitate their understanding of normal cellular processes including differentiation and mitosis as well as cancer pathologies.
