Ste5 RING-H2 Domain: Role in Ste4-Promoted Oligomerization for Yeast Pheromone Signaling

Abstract

Ste5 is a scaffold for the mitogen-activated protein kinase (MAPK) cascade components in a yeast pheromone response pathway. Ste5 also associates with Ste4, the beta subunit of a heterotrimeric guanine nucleotide-binding protein, potentially linking receptor activation to stimulation of the MAPK cascade. A RING-H2 motif at the Ste5 amino terminus is apparently essential for function because Ste5(C177S) and Ste5(C177A C180A) mutants did not rescue the mating defect of a ste5Delta cell. In vitro Ste5(C177A C180A) bound each component of the MAPK cascade, but not Ste4. Unlike wild-type Ste5, the mutant did not appear to oligomerize; however, when fused to a heterologous dimerization domain (glutathione S-transferase), the chimeric protein restored mating in an ste5Delta cell and an ste4Delta ste5Delta double mutant. Thus, the RING-H2 domain mediates Ste4-Ste5 interaction, which is a prerequisite for Ste5-Ste5 self-association and signaling.

The Bmi-1 oncoprotein interacts with dinG and MPh2: the role of RING finger domains

“…The Ste5 protein of budding yeast, a`sca old' protein that links signaling molecules of the pheromone response pathway, contains a RING-H2 domain (C 3 HHC 3 ). Mutations of the metal binding ligands block the ability of Ste5p to bind Ste4p but do not disturb the general structure or stablity of the protein (Inouye et al, 1997). Furthermore, the product of the familial breast cancer susceptibility gene BRCA1 contains a RING ®nger that is required for the interaction with another RING ®nger protein, BARD1.…”

“…BRCA1/BARD1 and Ste5p/Ste4p serve as additional examples in which the RING ®nger is required for protein-protein interactions (Wu et al, 1996;Inouye et al, 1997). Interestingly, BRCA1 and BARD1, like Bmi-1 and dinG, both contain RING ®ngers.…”

“…Alternatively, the RING ®nger may mediate protein-protein interactions. To date, there is evidence that the RING ®nger domain of human BRCA1 and yeast Ste5p are essential for speci®c protein interactions but there has been no de®nitive demonstration of speci®c DNA-RING ®nger binding (Wu et al, 1996;Inouye et al, 1997).…”

Nuclear Export and Plasma Membrane Recruitment of the Ste5 Scaffold Are Coordinated with Oligomerization and Association with Signal Transduction Components

“…It has been proposed that the association of Ste5 with Ste4 through the RING-H2 domain is a prerequisite for Ste5-Ste5 association and signaling (Inouye et al, 1997a). However, biochemical and two-hybrid analysis suggests that Ste5 oligomerization could occur constitutively (Feng et al, 1998).…”

“…On pheromone stimulation, the receptor activates a G protein by dissociating the G␣ (Gpa1) subunit from the G␤␥ dimer (Ste4/Ste18) (reviewed in Gustin et al, 1998). The G␤ subunit (Ste4) is then thought to bind to Ste5 Inouye et al, 1997a, Feng et al, 1998 and to Ste20 (Leeuw et al, 1998). This recruitment event is thought to allow Ste20 to directly phosphorylate Ste11 in the Ste5 scaffold complex (Feng et al, 1998;Pryciak and Huntress, 1998;van Drogen et al, 2000).…”