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Papers In Press, published online ahead of print November 26, 2001
Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017
Corresponding Author: kataoka{at}kobe-u.ac.jp
Mammalian candidate effectors of the small GTPase Ras, such as RalGDS, afadin/AF-6, Rin1 and phospholipase C
J. Biol. Chem, 10.1074/jbc.M109526200
Submitted on October 2, 2001
Revised on November 19, 2001
Accepted on November 26, 2001
Critical function of the Ras-associating domain as a primary Ras-binding site for regulation of Saccharomyces cerevisiae adenylyl cyclase
, have been shown to share structurally conserved modules termed Ras-associating (RA) domains at their Ras-binding sites. The Ras-binding domains of Raf-1 and phosphoinositide 3-kinase
, other Ras effectors, also share a similar tertiary structure with the RA domains. On the other hand, the primary Ras-binding site of Saccharomyces cerevisiae adenylyl cyclase, the best characterized Ras effector, has been mapped by mutational studies to the leucine-rich repeats (LRR) domain (amino acid 674-1300), whose structure apparently bears no resemblance to the RA domains. By a computer algorithm-based search we have unexpectedly found an RA domain in the N-terminal 81-amino acid residues (676-756) of the LRR domain. The purified RA-domain polypeptide exhibits an ability to bind directly to Ras in a GTP-dependent manner and to competitively inhibit Ras-dependent activation of adenylyl cyclase in vitro, with an affinity comparable to that of the whole LRR domain. The specificity of binding of the RA domain to various Ras effector region mutants is indistinguishable from that of the full-length adenylyl cyclase. The activated RAS2 (RAS2Val-19)-dependent heat shock sensitivity of yeast cells is suppressed by overexpression of the RA-domain polypeptide. Further, mutations of the RA domain abolish its Ras-binding activity, and adenylyl cyclase molecules carrying these mutations are rendered unactivatable by Ras in vitro. This RA domain bears highest similarity to the Ras-binding domain of Raf-1 based on comparison of its primary and predicted secondary structures with those of other Ras effectors. These results indicate that the RA domain is a primary Ras-binding site for activation of adenylyl cyclase, implicating RA domains as universal modules for interaction of effectors with Ras, conserved from yeast to mammals.
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