Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity.

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling.

During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] in the plasma membrane. Using fluorescently tagged high-affinity probes specific for PtdIns(4,5)P(2), we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)P(2), stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated PtdIns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)P(2) is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked self-reinforcing mechanism generates a spatially enriched pool of PtdIns(4,5)P(2) necessary for the membrane anchoring and function of a signaling complex.

Nucleus-specific and cell cycle-regulated degradation of mitogen-activated protein kinase scaffold protein Ste5 contributes to the control of signaling competence.

Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G(1) phase of the cell cycle. Ste5 scaffold protein is essential for pheromone response because it couples pheromone receptor stimulation to activation of the appropriate mitogen-activated protein kinase (MAPK) cascade. In naïve cells, Ste5 resides primarily in the nucleus. Upon pheromone treatment, Ste5 is rapidly exported from the nucleus and accumulates at the tip of the mating projection via its association with multiple plasma membrane-localized molecules. We found that concomitant with its nuclear export, the rate of Ste5 turnover is markedly reduced. Preventing nuclear export destabilized Ste5, whereas preventing nuclear entry stabilized Ste5, indicating that Ste5 degradation occurs mainly in the nucleus. This degradation is dependent on ubiquitin and the proteasome. We show that Ste5 ubiquitinylation is mediated by the SCF(Cdc4) ubiquitin ligase and requires phosphorylation by the G(1) cyclin-dependent protein kinase (cdk1). The inability to efficiently degrade Ste5 resulted in pathway activation and cell cycle arrest in the absence of pheromone. These findings reveal that maintenance of this MAPK scaffold at an appropriately low level depends on its compartment-specific and cell cycle-dependent degradation. Overall, this mechanism provides a novel means for helping to prevent inadvertent stimulus-independent activation of a response and for restricting and maximizing the signaling competence of the cell to a specific cell cycle stage, which likely works hand in hand with the demonstrated role that G(1) Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.

Function of the MAPK scaffold protein, Ste5, requires a cryptic PH domain.

Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered Gbetagamma freed upon pheromone receptor occupancy, thereby initiating downstream signaling. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic alpha-helix (PM motif) are not sufficient for Ste5 action. Rather, Ste5 contains a pleckstrin-homology (PH) domain (residues 388-518) that is essential for its membrane recruitment and function. Altering residues (R407S K411S) equivalent to those that mediate phosphoinositide binding in other PH domains abolishes Ste5 function. The isolated PH domain, but not a R407S K411S derivative, binds phosphoinositides in vitro. Ste5(R407S K411S) is expressed normally, retains Gbetagamma and Ste11 binding, and oligomerizes, yet is not recruited to the membrane in response to pheromone. Artificial membrane tethering of Ste5(R407S K411S) restores signaling. 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. 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).

Jekyll and Hyde in the microbial world.

Fungi are nonmotile organisms that obtain carbon from compounds in their immediate surroundings. Confronted with nutrient limitation, the yeast Saccharomyces cerevisiae undergoes a dimorphic transition, switching from spherical cells to filaments of adherent, elongated cells that can invade the substratum. A complex web of sensing mechanisms and cooperation among signaling networks (including a mitogen-activated protein kinase cascade, cyclic adenosine monophosphate-dependent protein kinase, and 5'-adenosine monophosphate-activated protein kinase) elicits the appropriate changes in physiology, cell cycle progression, cell polarity, and gene expression to achieve this differentiation. Highly related signaling processes control filamentation and virulence of many human fungal pathogens.