Editors' ChoicePheromone signaling

Formation of a Signaling Complex Through Synergistic Interactions

Science's STKE  18 Oct 2005:
Vol. 2005, Issue 306, pp. tw367
DOI: 10.1126/stke.3062005tw367

Ste5 is a scaffold that binds the mitogen-activated protein kinase (MAPK) module in yeast and is an adaptor connecting this MAPK module to the upstream regulatory elements Gβγ and the pheromone receptor and to the activating kinase PAK (Ste20), all of which are localized at the plasma membrane (PM). Ste5 is also localized to the PM. Winters et al. show that membrane localization of Ste5 is due to two synergistic interactions: one with the Gβγ subunit and one with acidic phospholipids at the inner leaflet of the PM mediated by a motif that likely forms an amphipathic α helix and also includes a nuclear localization signal (NLS). A Ste5 N-terminal (Ste5-NT) fragment containing the NLS and the Gβγ binding site localizes to the cell cortex and acts as a dominant-negative inhibitor of pheromone signaling. Deletion of the NLS disrupts the dominant-negative activity and cortical localization of this fragment without blocking Gβγ binding. A glutathione-S-transferase (GST) fusion protein with a membrane-binding fragment of Ste5 (residues 37 to 76) bound to liposomes in vitro, and in vivo the localization of the Ste5-NT fragment at the cortex was inhibited in cells deficient for the production of acidic phospholipids (specifically phosphoinositide 4-phosphate) at the PM. Mutational analysis of the PM-NLS sequence separated the nuclear localization activity from the PM localization activity and was consistent with the formation of an amphipathic α helix by this region of the protein and showed that nuclear localization was not required for the dominant-negative activity of the Ste5-NT fragment. The membrane-binding region of Ste5 was replaced with various other membrane-binding domains, such as pleckstrin homology (PH) domains or a basic-rich motif that binds phosphatidic acid-rich membranes, and these altered Ste5 proteins rescued pheromone signaling in yeast with a Ste5ΔNLS mutant. Analysis of various mutants of Ste5 that result in constitutive activation of the pheromone pathway indicated that these mutations increased membrane binding. Engineering of Ste5 mutants that had higher affinity binding for the membrane than did wild-type Ste5 resulted in a correlation between the activity of the signaling and the tendency of each mutant to bind the membrane. Thus, synergistic binding of Ste5 to Gβγ and the membrane through a relatively low-affinity membrane-binding motif allows for regulated activation of the pheromone pathway.

M. J. Winters, R. E. Lamson, H. Nakanishi, A. M. Neiman, P. M. Pryciak, A membrane binding domain in the Ste5 scaffold synergizes with Gβγ binding to control localization and signaling in pheromone response. Mol. Cell 20, 21-32 (2005). [PubMed]