We describe the identification and characterization of a novel gene,
sur-8 (suppressor of ras), that functions to regulate a receptor tyrosine kinase-Ras-MAP kinase-mediated signal transduction pathway during C. elegans vulval development. Two loss-of-function alleles of
sur-8 have been identified, one as a suppressor of the activated
let-60 ras (
n1046) mutation and a second in a non-complementation screen. Genetic analysis indicates that
sur-8 plays a positive regulatory role in Ras-mediated signaling and functions downstream of Ras but not downstream of Raf.
sur-8 cDNA expressed under the control of a heat shock promoter can rescue
sur-8 mutants only when they are heat shocked during L2 or L3, consistent with a requirement for
sur-8 activity during vulval induction.
sur-8 is predicted to encode a novel protein, conserved in mammals, that is composed predominantly of leucine-rich repeats (LRR). SUR-8 interacts with LET-60 Ras in the yeast two-hybrid system and in vitro. A missense mutation in a LRR, C233Y, encoded by the
sur-8(
ku242) allele, eliminates LET-60 Ras binding, indicating the importance of this LRR in both SUR-8 function and Ras binding. A putative LET-60 Ras effector domain mutation, P34G, also disrupts binding with SUR-8, but has no effect on binding with the Ras effector LIN-45 Raf. Conversley, a distinct effector domain mutation, E37G Y40C, disrupts interaction with LIN-45 Raf but not with SUR-8. Thus SUR-8 binding requires effector domain residues that are distinct from those required by Raf for Ras binding. In addition to disrupting SUR-8 interaction, P34G has been shown to disrupt the binding of another LRR containing effector protein, yeast adenylate cyclase. Thus, LRR mediated interaction with Ras may be an evolutionarily conserved mechanism for effector interactions. However, while interaction between Ras and yeast adenylate cyclase is GTP dependent, in vitro binding of SUR-8 is not GTP dependent since SUR-8 binds Ras-GTP with the same affinity as Ras-GDP. The human and mouse SUR-8 homologues are 57% identical to Ce SUR-8 and have the same number and organization of LRRs. Human
sur-8 coding region under the control of a heat shock promoter rescues
sur-8 mutants, and human SUR-8 binds LET-60 Ras in vitro, indicating a conserved function as well as structure. In addition, human SUR-8 interacts with human K-Ras and N-Ras but not with H-Ras in the yeast two-hybrid system and in vitro. Our results indicate that
sur-8 is an evolutionarily conserved positive regulator of Ras signaling pathways and that SUR-8 may mediate its effects through binding specific Ras family members.