![]() Bem1, an SH3 domain-containing adaptor protein, has an internal Phox-homology (PX) domain, and PX domains can bind phosphoinositides ( Sato et al. 1996) and, most recently, Cdc24 ( Wang et al. Interactions of Ste5 with other plasma membrane-associated proteins, including Bem1 ( Leeuw et al. Perhaps the mutants are secured at the cell cortex, even in the absence of Gβγ, because they enhance Ste5 association with other proteins that are themselves membrane-bound in a Gβγ-independent manner. This quandary suggested that the mutants unveil an alternative, relatively efficient, Gβγ-independent means by which Ste5 associates with the membrane. Our ability to isolate such alleles raised a paradox, given the evidence that Ste5–Gβγ association is critical for membrane delivery of Ste11 and its activation by Ste20. Although these alleles bypass the need for Gβγ, they do not bypass the need for the other known components of the pathway, most notably Ste20. 2000) by selecting for alterations of Ste5 that permit signaling even in cells lacking Gβγ. To further delineate how Ste5 contributes to signal propagation, we isolated three mutations (P44L, C226Y, S770N) that constitutively activate Ste5 ( Sette et al. 1999), and reimport of Ste5 into the nucleus contributes to down-regulation of signaling ( Künzler et al. Ste5 undergoes constitutive nucleocytoplasmic shuttling in naïve cells, but accumulates stably at the cell cortex in the projection (“shmoo tip”) that forms on pheromone-treated cells ( Pryciak and Huntress 1998 Mahanty et al. Ste5 self-associates, and this oligomerization is important for efficient signaling ( Yablonski et al. Thus, like Ste5, Ste20 concentrates near activated pheromone receptors. Activated Ste20 is membrane-associated not only by binding of its N-terminal CRIB domain to Cdc42-GTP, but also because its C terminus contains a high-affinity binding site for Gβγ ( Leeuw et al. 2002), which is also membrane-anchored via C-terminal lipophilic modifications ( Ohya et al. The p21 that activates Ste20 is the small GTPase, Cdc42 ( Lamson et al. 2000), a so-called p21-activated protein kinase (PAK). Gβγ-dependent docking of Ste5 at the plasma membrane permits efficient encounter of Ste5-bound Ste11 with its activator (MAPKKKK), Ste20 ( Drogen et al. Gβγ remains tethered at the plasma membrane via lipophilic modifications at the C-terminal end of Gγ (Ste18) ( Hirschman and Jenness 1999). The region of Ste5 responsible for interaction with Gβγ includes a RING-H2 domain near its N terminus ( Inouye et al. Second, Ste5 delivers its kinase cargo to the plasma membrane, at least in part, via association with the Gβ subunit (Ste4) of the Gβγ complex released from the receptor-associated heterotrimeric G-protein ( Whiteway et al. 1997a) thus, Ste5 was the first recognized MAPK scaffold protein. 1994 Printen and Sprague 1994 Inouye et al. Distinct regions of Ste5 interact with each kinase, as delimited by mutational analysis ( Choi et al. Ste5 protein ( Elion 2001) is essential for this coupling: First, because it binds the MAPKKK (Ste11), the MAPKK (Ste7), and the MAPK (Fus3) of the MAPK cascade. Yeast pheromone response links activation of a G-protein-coupled receptor to stimulation of an appropriate mitogen-activated protein kinase (MAPK) (for review, see Wang and Dohlman 2004). Thus, the PH domain is essential for stable membrane recruitment of Ste5, and this association is critical for initiation of downstream signaling because it allows Ste5-bound Ste11 (MAPKKK) to be activated by membrane-bound Ste20 (MAPKKKK). R407S K411S loss-of-function mutations abrogate the constitutive activity of gain-of-function Ste5 alleles, including one (P44L) that increases membrane affinity of the PM motif. Artificial membrane tethering of Ste5(R407S K411S) restores signaling. Ste5(R407S K411S) is expressed normally, retains Gβγ and Ste11 binding, and oligomerizes, yet is not recruited to the membrane in response to pheromone. The isolated PH domain, but not a R407S K411S derivative, binds phosphoinositides in vitro. Altering residues (R407S K411S) equivalent to those that mediate phosphoinositide binding in other PH domains abolishes Ste5 function. Rather, Ste5 contains a pleckstrin-homology (PH) domain (residues 388–518) that is essential for its membrane recruitment and function. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic α-helix (PM motif) are not sufficient for Ste5 action. Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered Gβγ freed upon pheromone receptor occupancy, thereby initiating downstream signaling.
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