Professor of Biochemistry, Biophysics and Structural Biology*
*And William V. Power Chair in Biology (1991-2011), And Affiliate, Division of Cell and Developmental Biology
Transmembrane and intracellular signal transduction mechanisms are the focus of our group, especially understanding how extracellular stimuli control cell growth and division, cell morphology, and gene expression at the biochemical level.
Structural analysis of a peptide hormone receptor. To understand the molecular biology of peptide hormone action, we study response of budding yeast (Saccharomyces cerevisiae) to its peptide mating pheromones (a-factor and α-factor). The pheromone receptors have seven hydrophobic segments and are coupled to a heterotrimeric G protein. Receptors of this type are ubiquitous and transduce binding of a wide variety of extracellular ligands (peptide hormones, neurotransmitters and other bioactive compounds) into a physiological signal. Attempts to determine the three-dimensional structure of the purified α-factor receptor by NMR and X-ray crystallography are underway in collaborative studies. We are also interested in proteins involved in adaptation and recovery after the pheromone-induced signal, especially: structure and function of Sst2, the prototype RGS (Regulator of G-protein Signaling) protein; α-arrestins involved in selective ubiquitinylation and endocytosis of integral membrane proteins; and, various classes of phosphotyrosine-directed, phosphoserine- / phosphothreonine-directed, and dual-specificity phosphoprotein phosphatases that can act to dephosphorylate activated MAPKs.
Molecular genetics and biochemistry of a protein kinase cascade. Activation of the receptor-coupled G protein initiates a cascade of three protein kinases, resulting in stimulation of a messenger-activated protein kinase (MAPK) in the nucleus. A different developmental pathway (invasive or filamentous growth) triggered by nutrient limitation stimulates another MAPK. Subjecting cells to hyperosmotic conditions activates yet another MAPK. Thus, MAPK cascades are universally employed for signal transduction in eukaryotic cells, and every eukaryotic cell contains multiple MAPK pathways. We are investigating the mechanisms that impose specificity and fidelity at each tier in these signaling networks. We showed that one device used by the cell for discrimination between parallel MAPK pathways is a specific docking interaction between a MAPK and the N-terminus of its cognate upstream protein kinase (MEK). We also showed that a scaffold protein, Ste5, helps ensure signaling fidelity in pheromone response by binding the appropriate MAPK, MEK, and upstream activating kinase (MEKK) and by shuttling from the nucleus to the plasma membrane and delivering the MAPK module to its most proximal activator (a fourth membrane-associated protein kinase). A different scaffold protein, Ste50, is required for signal propagation in the invasive growth and hyperosmotic stress response pathways and is also under study. The roles of other factors that prevent inappropriate MAPK activation in response to a given stimulus are also under study.
Control of gene expression. We used genetic and biochemical methods to demonstrate that two negative transcriptional regulators are substrates of the MAPKs. How the function of these targets is modified by phosphorylation is under study. We also discovered a general regulator of transcription, which has homologs in Drosophila and human cells, that catalyzes ATP-dependent dissociation of TATA box-binding protein (TBP)-DNA complexes. Structural analysis of the mechanism of action of this global transcriptional regulator is in progress.
Protein kinases involved in cell proliferation, differentiation, and cell cycle control. The Wee1 class of protein-tyrosine kinase has an important role in cell cycle control. We are investigating pathways that regulate the activity, localization, and stability of this enzyme, including its recruitment to septin filaments, which assemble at the presumptive site of cell division. We have shown that septin filaments are assembled from hetero-octameric complexes containing two each of four different septin subunits. A considerable effort is underway in the lab to understand the regulation of septin complex assembly, the formation, supramolecular architecture and disassembly of septin filaments, and the function of septin filaments in the events required for cell division and membrane septation during cytokinesis. A cascade of protein kinases conserved from yeast to humans, including the TORC2 complex, that is involved in maintenance of sphingolipid and glycerolipid homeostasis in the plasma membrane is also under intensive study.
Molecular biology of phosphoinositide-dependent signaling. In previous work, we have shown that phosphatidylinositol 4-phosphate generated by a specific phosphatidylinositol 4-kinase isoform has a specific role in the Golgi-to-plasma membrane stage of the secretory pathway. This enzyme also has an essential role in supplying the PtdIns(4)P that is converted to PtdIns(4,5)P2 in the nucleus, where it is hydrolyzed by a specific phospholipase C to generate inositol polyphosphates that regulate transcription, chromatin remodeling and mRNA export. We found that the phosphatidylinositol 4-kinase is also regulated by a small calcium-binding protein whose ortholog in animal cells (frequenin or neuronal calcium sensor-1) is found mainly in neuronal and neuroendocrine cells.
Novel mechanisms for translocation across membranes. We discovered that export of a-factor pheromone requires an integral plasma membrane protein that is a dedicated ATP-dependent transporter, rather than the classical secretory pathway. The mechanism of transmembrane translocation of pheromone by the transporter (Ste6) is being examined. The functions of other members of the same transporter class, namely ABC transporters, are also under study.
Konopka JB, Thorner JW (2012) "Pheromone receptors (yeast)." In Encyclopedia of Biological Chemistry, 2nd Ed. (Lane MD, Lennarz WJ, eds.), Elsevier Science, Inc., Oxford, UK, in press.
Bertin A, McMurray MA, Pierson J, Thai L, McDonald KL, Zehr EA, Garcia G 3rd, Peters P, Thorner J, Nogales E (2012) Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiae. Mol. Biol. Cell 23: 423-432.
Roelants FM, Breslow DK, Muir A, Weissman JS, Thorner J (2011) Protein kinase Ypk1 phosphorylates Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 108: 19222-19227.
Garcia G III, Bertin A, Li Z, Song Y, McMurray MA, Thorner J, Nogales E (2011) Subunit-dependent modulation of septin assembly: Budding yeast septin Shs1 promotes ring and gauze formation. J. Cell Biol. 195: 993-1004.
McMurray MA, Stefan CJ, Wemmer M, Odorizzi G, Emr SD, Thorner J (2011) Genetic interactions with mutations affecting septin assembly reveal ESCRT functions in budding yeast cytokinesis. Biol. Chem. 392: 699-712.
McMurray MA, Bertin A, Garcia G 3rd, Lam L, Nogales E, Thorner J (2011) Septin filament formation is essential in budding yeast. Dev. Cell 20: 540-549.
Lim S, Strahl T, Thorner J, Ames JB (2011) Structure of a Ca2+-myristoyl switch protein that controls activation of a phosphatidylinositol 4-kinase in fission yeast. J. Biol. Chem. 286: 12565-12577.
Patterson JC, Klimenko ES, Thorner J (2010) Single-cell analysis reveals that insulation maintains signaling specificity between two yeast MAPK pathways with common components. Sci. Sig. 3: ra75.1-ra75.11.
Bertin A, McMurray MA, Thai L, Garcia G III, Votin V, Grob P, Allyn T, Thorner J, Nogales E (2010) Phosphatidylinositol-4,5-bisphosphate promotes budding yeast septin filament assembly and organization. J. Mol. Biol. 404: 711-731.
Chen RE, Patterson JC, Goupil LS, Thorner J (2010) Dynamic localization of Fus3 MAPK is necessary to evoke appropriate responses and avoid cytotoxic effects. Mol. Cell. Biol. 30: 4293-4307.
Chen RE, Thorner J (2010) Systematic epistasis analysis of the contributions of PKA- and MAPK-dependent signaling to nutrient limitation-evoked responses in the yeast Saccharomyces cerevisiae. Genetics 185: 855-870.
Garrenton LS, Stefan CJ, McMurray MA, Emr SD, Thorner J (2010) Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling. Proc. Natl. Acad. Sci. USA 107: 11805-11810.
Roelants FM, Baltz AG, Trott AE, Fereres S, Thorner J (2010) A protein kinase network regulates the function of aminophospholipid flippases. Proc. Natl. Acad. Sci. USA 107: 34-39.
Garrenton LS, Braunwarth A, Irniger S, Hurt E, Künzler M, Thorner J (2009) Nucleus-specific and cell cycle-regulated degradation of mitogen-activated protein kinase scaffold protein Ste5 contributes to the control of signaling competence. Mol. Cell. Biol. 29: 582-601.
McMurray MA, Thorner J (2009) Septins: molecular partitioning and the generation of cellular asymmetry. Cell Div. 4: 18.1-18.40.
McMurray MA, Thorner J (2009) Reuse, replace, recycle. Specificity in subunit inheritance and assembly of higher-order septin structures during mitotic and meiotic division in budding yeast. Cell Cycle 8: 195-203.
Rockwell NC, Wolfger H, Kuchler K, Thorner J (2009) ABC transporter Pdr10 regulates the membrane microenvironment of Pdr12 in Saccharomyces cerevisiae. J. Membr. Biol. 229: 27-52.
Westfall PJ, Patterson JC, Chen RE, Thorner J (2008) Stress resistance and signal fidelity independent of nuclear MAPK function. Proc. Natl. Acad. Sci. USA 105: 12212-12217.
McMurray MA, Thorner J (2008) Septin stability and recycling during dynamic structural transitions in cell division and development. Curr. Biol. 18: 1203-1208.
McMurray MA, Thorner J (2008) Biochemical properties and supramolecular architecture of septin hetero-oligomers and septin filaments, In "The Septins" (Hall PA, Russell SEG, Pringle JR, Eds.), John Wiley & Sons, Ltd., Chicester, West Sussex, UK, pp. 49-100.
Bertin A, McMurray MA, Grob P, Park SS, Garcia G 3rd, Patanwala I, Ng HL, Alber T, Thorner J, Nogales E (2008) Saccharomyces cerevisiae septins: supramolecular organization of heterooligomers and the mechanism of filament assembly. Proc. Natl. Acad. Sci. USA 105: 8274-8279.
Last Updated 2012-08-14