| Research Abstract |
Several different types of GTP-dependent Ras-binding domains have been identified so far for the GAP/NF1 family, the Raf family, PI3K,RalGDS,and so on, but no apparent sequence homology have been elucidated for them. It is expected that a mutant Ras protein with a binding specificity restricted to a subset of those target proteins is a useful tool for analysis of the complicated Ras signaling pathways. In this study, we made Ha-Ras mutants carrying substitution (s) in the region of residues 21-71, and tested their abilities for association with different Ras-binding domains. Actually, we found several mutants that bind to a restricted range of targets. An NMR analysis showed that some of these mutants do not exhibit the "regional polysterism", which has been observed for the GTP-bound from of the wild-type Ras protein. Therefore, we propose that the "regional polysterism" of Ras allows the binding to various target molecules. In fact, we found that the Ras protein takes only a single conformation in a complex with the "Ras-binding domain (RBD) " of Raf-1 or of RGL.
Some mutations of Ras within its "activator region" whose conformations are unaffected by GDP/GTP exchenge, were found to be involved in the binding to another Ras-binding domain, the "Cry-rich domain (CRD), " of Raf-1. Furthermore, these mutations in the activator regions of Ras were found to impair the ability of Ras to activate the Raf-1 kinase activity. Thus, the Raf-1 activation is found to require Res to bind to both RBD and CRD.Furthermore, replacement of the reside (Glu) at position 31 of Ras with Lys increased the Raf-1 CRD binding activity abnormally and inhibited the Raf-1 activation ability of Ras.
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