Biomedical subjects

Jeremy Thorner

Publications and source records attributed to Jeremy Thorner.

At least 19 recordsLinked to original sources

PubMed2006

DEP-domain-mediated regulation of GPCR signaling responses.

G protein-coupled receptors (GPCRs) mediate cellular responses to a variety of stimuli, but how specific responses are regulated has been elusive, as the types of GPCRs vastly outnumber the classes of G protein heterotrimers available to initiate downstream signaling. In our analysis of signaling proteins containing DEP domains ( approximately 90 residue sequence motifs first recognized in fly Dishevelled, worm EGL-10, and mammalian Pleckstrin), we find that DEP domains are responsible for specific recognition of GPCRs. We examined the yeast regulator of G protein signaling (RGS) protein Sst2 and demonstrate that the DEP domains in Sst2 mediate binding to its cognate GPCR (Ste2). DEP-domain-mediated tethering promotes downregulation by placing the RGS protein in proximity to its substrate (receptor-activated Galpha subunit). Sst2 docks to the Ste2 cytosolic tail, but only its unphosphorylated state, allowing for release and recycling of this regulator upon receptor desensitization and internalization. DEP-domain-mediated targeting of effectors and regulators to specific GPCRs provides a means to dictate the nature, duration, and specificity of the response.

Amino Acid Sequence↗

PubMed2006

Direct phosphorylation and activation of a Nim1-related kinase Gin4 by Elm1 in budding yeast.

In budding yeast, Gin4, a Nim1-related kinase, plays an important role in proper organization of the septin ring at the mother-bud neck, a filamentous structure that is critical for diverse cellular processes including mitotic entry and cytokinesis. How Gin4 kinase activity is regulated is not known. Here we showed that a neck-associated Ser/Thr kinase Elm1, which is important for septin assembly, is critical for proper modification of Gin4 and its physiological substrate Shs1. In vitro studies with purified recombinant proteins demonstrated that Elm1 directly phosphorylates and activates Gin4, which in turn phosphorylates Shs1. Consistent with these observations, acute inhibition of Elm1 activity abolished mitotic Gin4 phosphorylation and Gin4-dependent Shs1 modification in vivo. In addition, a gin4 mutant lacking the Elm1-dependent phosphorylation sites exhibited an impaired localization to the bud-neck and, as a result, induced a significant growth defect with an elongated bud morphology. Thus, Elm1 regulates the septin assembly-dependent cellular events by directly phosphorylating and activating the Gin4-dependent pathway(s).

Animals↗

PubMed2006

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).

Adaptor Proteins, Signal Transducing↗

PubMed2006

Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its coactivator InsP6 is required for mRNA export.

The DExD/H-box ATPase Dbp5 is essential for nuclear mRNA export, although its precise role in this process remains poorly understood. Here, we identify the nuclear pore protein Gle1 as a cellular activator of Dbp5. Dbp5 alone is unable to stably bind RNA or effectively hydrolyse ATP under physiological conditions, but addition of Gle1 dramatically stimulates these activities. A gle1 point mutant deficient for Dbp5 stimulation in vitro displays an mRNA export defect in vivo, indicating that activation of Dbp5 is an essential function of Gle1. Interestingly, Gle1 binds directly to inositol hexakisphosphate (InsP6) and InsP6 potentiates the Gle1-mediated stimulation of Dbp5. Dominant mutations in DBP5 and GLE1 that rescue mRNA export phenotypes associated with the lack of InsP6 mimic the InsP6 effects in vitro. Our results define specific functions for Gle1 and InsP6 in mRNA export and suggest that local activation of Dbp5 at the nuclear pore is critical for mRNA export.

Active Transport, Cell Nucleus↗

PubMed2006

Analysis of mitogen-activated protein kinase signaling specificity in response to hyperosmotic stress: use of an analog-sensitive HOG1 allele.

When confronted with a marked increase in external osmolarity, budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high-osmolarity glycerol or HOG pathway) to elicit cellular responses necessary to permit continued growth. One input that stimulates the HOG pathway requires the integral membrane protein and putative osmosensor Sho1, which recruits and enables activation of the MAPK kinase kinase Ste11. In mutants that lack the downstream MAPK kinase (pbs2Delta) or the MAPK (hog1Delta) of the HOG pathway, Ste11 activated by hyperosmotic stress is able to inappropriately stimulate the pheromone response pathway. This loss of signaling specificity is known as cross talk. To determine whether it is the Hog1 polypeptide per se or its kinase activity that is necessary to prevent cross talk, we constructed a fully functional analog-sensitive allele of HOG1 to permit acute inhibition of this enzyme without other detectable perturbations of the cell. We found that the catalytic activity of Hog1 is required continuously to prevent cross talk between the HOG pathway and both the pheromone response and invasive growth pathways. Moreover, contrary to previous reports, we found that the kinase activity of Hog1 is necessary for its stress-induced nuclear import. Finally, our results demonstrate a role for active Hog1 in maintaining signaling specificity under conditions of persistently high external osmolarity.

Alleles↗

PubMed2006

The RA domain of Ste50 adaptor protein is required for delivery of Ste11 to the plasma membrane in the filamentous growth signaling pathway of the yeast Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, pheromone response requires Ste5 scaffold protein, which ensures efficient G-protein-dependent recruitment of mitogen-activated protein kinase (MAPK) cascade components Ste11 (MAPK kinase kinase), Ste7 (MAPK kinase), and Fus3 (MAPK) to the plasma membrane for activation by Ste20 protein kinase. Ste20, which phosphorylates Ste11 to initiate signaling, is activated by binding to Cdc42 GTPase (membrane anchored via its C-terminal geranylgeranylation). Less clear is how activated and membrane-localized Ste20 contacts Ste11 to trigger invasive growth signaling, which also requires Ste7 and the MAPK Kss1, but not Ste5. Ste50 protein associates constitutively via an N-terminal sterile-alpha motif domain with Ste11, and this interaction is required for optimal invasive growth and hyperosmotic stress (high-osmolarity glycerol [HOG]) signaling but has a lesser role in pheromone response. We show that a conserved C-terminal, so-called "Ras association" (RA) domain in Ste50 is also essential for invasive growth and HOG signaling in vivo. In vitro the Ste50 RA domain is not able to associate with Ras2, but it does associate with Cdc42 and binds to a different face than does Ste20. RA domain function can be replaced by the nine C-terminal, plasma membrane-targeting residues (KKSKKCAIL) of Cdc42, and membrane-targeted Ste50 also suppresses the signaling deficiency of cdc42 alleles specifically defective in invasive growth. Thus, Ste50 serves as an adaptor to tether Ste11 to the plasma membrane and can do so via association with Cdc42, thereby permitting the encounter of Ste11 with activated Ste20.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Yeast phosphatidylinositol 4-kinase, Pik1, has essential roles at the Golgi and in the nucleus.

Phosphatidylinositol 4-kinase, Pik1, is essential for viability. GFP-Pik1 localized to cytoplasmic puncta and the nucleus. The puncta colocalized with Sec7-DsRed, a marker of trans-Golgi cisternae. Kap95 (importin-beta) was necessary for nuclear entry, but not Kap60 (importin-alpha), and exportin Msn5 was required for nuclear exit. Frq1 (frequenin orthologue) also is essential for viability and binds near the NH2 terminus of Pik1. Frq1-GFP localized to Golgi puncta, and Pik1 lacking its Frq1-binding site (or Pik1 overexpressed in frq1Delta cells) did not decorate the Golgi, but nuclear localization was unperturbed. Pik1(Delta10-192), which lacks its nuclear export sequence, displayed prominent nuclear accumulation and did not rescue inviability of pik1Delta cells. A Pik1-CCAAX chimera was excluded from the nucleus and also did not rescue inviability of pik1Delta cells. However, coexpression of Pik1(Delta10-192) and Pik1-CCAAX in pik1Delta cells restored viability. Catalytically inactive derivatives of these compartment-restricted Pik1 constructs indicated that PtdIns4P must be generated both in the nucleus and at the Golgi for normal cell function.

1-Phosphatidylinositol 4-Kinase↗

PubMed2005

Systems biology approaches in cell signaling research.

The use of methods for global and quantitative analysis of cells is providing new systems-level insights into signal transduction processes. Recent studies reveal important information about the rates of signal transmission and propagation, help establish some general regulatory characteristics of multi-tiered signaling cascades, and illuminate the combinatorial nature of signaling specificity in cell differentiation.

Animals↗

PubMed2005

Reconstitution of the mammalian PI3K/PTEN/Akt pathway in yeast.

The mammalian signalling pathway involving class I PI3K (phosphoinositide 3-kinase), PTEN (phosphatidylinositol 3-phosphatase) and PKB (protein kinase B)/c-Akt has roles in multiple processes, including cell proliferation and apoptosis. To facilitate novel approaches for genetic, molecular and pharmacological analyses of these proteins, we have reconstituted this signalling pathway by heterologous expression in the unicellular eukaryote, Saccharomyces cerevisiae (yeast). High-level expression of the p110 catalytic subunit of mammalian PI3K dramatically inhibits yeast cell growth. This effect depends on PI3K kinase activity and is reversed partially by a PI3K inhibitor (LY294002) and reversed fully by co-expression of catalytically active PTEN (but not its purported yeast orthologue, Tep1). Growth arrest by PI3K correlates with loss of PIP2 (phosphatidylinositol 4,5-bisphosphate) and its conversion into PIP3 (phosphatidylinositol 3,4,5-trisphosphate). PIP2 depletion causes severe rearrangements of actin and septin architecture, defects in secretion and endocytosis, and activation of the mitogen-activated protein kinase, Slt2. In yeast producing PIP3, PKB/c-Akt localizes to the plasma membrane and its phosphorylation is enhanced. Phospho-specific antibodies show that both active and kinase-dead PKB/c-Akt are phosphorylated at Thr308 and Ser473. Thr308 phosphorylation, but not Ser473 phosphorylation, requires the yeast orthologues of mammalian PDK1 (3-phosphoinositide-dependent protein kinase-1): Pkh1 and Pkh2. Elimination of yeast Tor1 and Tor2 function, or of the related kinases (Tel1, Mec1 and Tra1), did not block Ser473 phosphorylation, implicating another kinase(s). Reconstruction of the PI3K/PTEN/Akt pathway in yeast permits incisive study of these enzymes and analysis of their functional interactions in a simplified context, establishes a new tool to screen for novel agonists and antagonists and provides a method to deplete PIP2 uniquely in the yeast cell.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2005

Some assembly required: yeast septins provide the instruction manual.

Septins are a family of conserved proteins that form hetero-oligomeric complexes that assemble into filaments. The filaments can be organized into linear arrays, coils, rings and gauzes. They serve as membrane-associated scaffolds and as barriers to demarcate local compartments, especially for the establishment of the septation site for cytokinesis. Studies in budding and fission yeast have revealed many of the protein-protein interactions that govern the formation of multi-septin complexes. GTP binding and phosphorylation direct the polymerization of filaments that is required for septin-collar assembly in budding yeast, whereas a homolog of anillin instructs timely formation of the ring of septin filaments at the medial cortex in fission yeast. These insights should aid understanding of the organization and function of the diverse septin structures in animal cells.

Animals↗

PubMed2004

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.

AMP-Activated Protein Kinases↗

PubMed2004

When the stress of your environment makes you go HOG wild.

When exposed to increased dissolved solute in their environment (hyperosmotic stress), all eukaryotic cells respond by rapidly activating a conserved mitogen-activated protein kinase cascade, known in budding yeast Saccharomyces cerevisiae as the high osmolarity glycerol (HOG) pathway. Intensive genetic and biochemical analysis in this organism has revealed the presumptive osmosensors, downstream signaling components, and metabolic and transcriptional changes that allow cells to cope with this stressful condition. These findings have had direct application to understanding stress sensing and control of transcription by stress-activated mitogen-activated protein kinases in mammalian cells.

Cell Nucleus↗

PubMed2004

Protein-protein interactions governing septin heteropentamer assembly and septin filament organization in Saccharomyces cerevisiae.

Mitotic yeast (Saccharomyces cerevisiae) cells express five related septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) that form a cortical filamentous collar at the mother-bud neck necessary for normal morphogenesis and cytokinesis. All five possess an N-terminal GTPase domain and, except for Cdc10, a C-terminal extension (CTE) containing a predicted coiled coil. Here, we show that the CTEs of Cdc3 and Cdc12 are essential for their association and for the function of both septins in vivo. Cdc10 interacts with a Cdc3-Cdc12 complex independently of the CTE of either protein. In contrast to Cdc3 and Cdc12, the Cdc11 CTE, which recruits the nonessential septin Shs1, is dispensable for its function in vivo. In addition, Cdc11 forms a stoichiometric complex with Cdc12, independent of its CTE. Reconstitution of various multiseptin complexes and electron microscopic analysis reveal that Cdc3, Cdc11, and Cdc12 are all necessary and sufficient for septin filament formation, and presence of Cdc10 causes filament pairing. These data provide novel insights about the connectivity among the five individual septins in functional septin heteropentamers and the organization of septin filaments.

Amino Acid Sequence↗

PubMed2004

Coupling morphogenesis to mitotic entry.

In eukaryotes, cyclin B-bound cyclin-dependent protein kinase 1 promotes mitotic entry but is held in check, in part, by Wee1 protein kinase. Timely mitotic entry in budding yeast requires inactivation of Swe1 (Wee1 ortholog). Perturbations of the septin collar at the bud neck lead to Swe1 stabilization, delaying the G(2)/M transition. Swe1 is recruited to the neck and hyperphosphorylated before ubiquitin-mediated degradation. Hsl1 kinase (Nim1 ortholog), a negative regulator of Wee1, is required for efficient Swe1 localization at the neck but seems not to phosphorylate Swe1. Here, we show that two other kinases targeted sequentially to the neck, Cla4/PAK and Cdc5/Polo, are responsible for stepwise phosphorylation and down-regulation of Swe1. This mechanism links assembly of a cellular structure to passage into mitosis.

Cell Cycle Proteins↗

PubMed2004

Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4.

Assembly at the mother-bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis. We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro. In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not. GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro. Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation. This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression. Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo. Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.

Binding Sites↗

PubMed2004

Differential roles of PDK1- and PDK2-phosphorylation sites in the yeast AGC kinases Ypk1, Pkc1 and Sch9.

Saccharomyces cerevisiae Pkh1 and Pkh2 (orthologues of mammalian protein kinase, PDK1) are functionally redundant. These kinases activate three AGC family kinases involved in the maintenance of cell wall integrity: Ypk1 and Ypk2, two closely related, functionally redundant enzymes (orthologues of mammalian protein kinase SGK), and Pkc1 (orthologue of mammalian protein kinase PRK2). Pkh1 and Pkh2 activate Ypk1, Ypk2 and Pkc1 by phosphorylating a Thr in a conserved sequence motif (PDK1 site) within the activation loop of these proteins. A fourth protein kinase involved in growth control and stress response, Sch9 (orthologue of mammalian protein kinase c-Akt/PKB), also carries the conserved activation loop motif. Like other AGC family kinases, Ypk1, Ypk2, Pkc1 and Sch9 also carry a second conserved sequence motif situated in a region C-terminal to the catalytic domain, called the hydrophobic motif (PDK2 site). Currently, there is still controversy surrounding the identity of the enzyme responsible for phosphorylating this second site and the necessity for phosphorylation at this site for in vivo function. Here, genetic and biochemical methods have been used to investigate the physiological consequences of phosphorylation at the PDK1 and PDK2 sites of Ypk1, Pkc1 and Sch9. It was found that phosphorylation at the PDK1 site in the activation loop is indispensable for the essential functions of all three kinases in vivo, whereas phosphorylation at the PDK2 motif plays a non-essential and much more subtle role in modulating the ability of these kinases to regulate the downstream processes in which they participate.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2003

Roles of phosphoinositides and of Spo14p (phospholipase D)-generated phosphatidic acid during yeast sporulation.

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P2 synthesis.

1-Butanol↗

PubMed2003

Conservation of regulatory function in calcium-binding proteins: human frequenin (neuronal calcium sensor-1) associates productively with yeast phosphatidylinositol 4-kinase isoform, Pik1.

Frequenin, also known as neuronal calcium sensor-1 (NCS-1), is an N-myristoylated Ca2+-binding protein that has been conserved in both sequence and three-dimensional fold during evolution. We demonstrate using both genetic and biochemical approaches that the observed structural conservation between Saccharomyces cerevisiae frequenin (Frq1) and human NCS-1 is also reflected at the functional level. In yeast, the sole essential target of Frq1 is the phosphatidylinositol 4-kinase isoform, Pik1; both FRQ1 and PIK1 are indispensable for cell viability. Expression of human NCS-1 in yeast, but not a close relative (human KChIP2), rescues the inviability of frq1 cells. Furthermore, in vitro, Frq1 and NCS-1 (either N-myristoylated or unmyristoylated) compete for binding to a small 28-residue motif near the N terminus of Pik1. Site-directed mutagenesis indicates that the binding determinant in Pik1 is a hydrophobic alpha-helix and that frequenins bind to one side of this alpha-helix. We propose, therefore, that the function of NCS-1 in mammals may closely resemble that of Frq1 in S. cerevisiae and, hence, that frequenins in general may serve as regulators of certain isoforms of phosphatidylinositol 4-kinase.

1-Phosphatidylinositol 4-Kinase↗