title: Function of the MAPK scaffold protein, Ste5, requires a cryptic PH domain.
abstract: 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).
authors.affiliation: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, 94720, USA.
authors.forename: Lindsay S
authors.initials: LS
authors.lastname: Garrenton
authors.forename: Susan L
authors.initials: SL
authors.lastname: Young
authors.forename: Jeremy
authors.initials: J
authors.lastname: Thorner
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010675:Pheromones / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D047908:Intracellular Signaling Peptides and Proteins; D010675:Pheromones; D010716:Phosphatidylinositols; D010750:Phosphoproteins; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C039077:platelet protein P47; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C078754:STE20 protein, S cerevisiae; C067018:Ste11 protein, S cerevisiae