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Biomedical subjects

J Thorner

Publications and source records attributed to J Thorner.

At least 37 records · Page 2Linked to original sources
PubMed1997

Casein kinase II catalyzes tyrosine phosphorylation of the yeast nucleolar immunophilin Fpr3.

In the yeast Saccharomyces cerevisiae, the nucleolar immunophilin, Fpr3, is phosphorylated at tyrosine and dephosphorylated by the phosphotyrosine-specific phosphoprotein phosphatase, Ptp1. In Ptp1-deficient cells, Fpr3 contains phospho-Tyr at a single site (Tyr184), but also contains phospho-Ser and phospho-Thr. Ser186 (adjacent to Tyr184) is situated within a canonical site for phosphorylation by casein kinase II (CKII). Yeast cell lysates contain an activity that binds to Fpr3 in vitro and phosphorylates Fpr3 at Ser, Thr, and Tyr; this activity was found to be dependent on expression of functional yeast CKII. Moreover, purified Fpr3 was phosphorylated on Tyr184 in vitro by either purified yeast CKII or purified, bacterially-expressed human CKII. Likewise, phosphorylation of Fpr3 at tyrosine in vivo was markedly enhanced in yeast cells overexpressing a heterologous (Drosophila) CKII, but was undetectable in yeast cells carrying only a temperature-sensitive allele of the endogenous CKII, even when the cells were grown at a permissive temperature. Phosphorylation of Fpr3 at Tyr184 by CKII in vitro lagged behind phosphorylation of Fpr3 at Ser, and was accelerated by pre-phosphorylation of Fpr3 at Ser using CKII. Furthermore, synthetic peptides corresponding to the sequence surrounding Tyr184 that contained P-Ser (or Glu) at position 186 were much more efficient substrates for CKII phosphorylation of Tyr184 than a synthetic peptide containing Ala at position 186. These findings indicate that CKII phosphorylates Fpr3 at tyrosine and serine both in vivo and in vitro and thus possesses dual specificity. These results also indicate that Tyr184 is phosphorylated by CKII via a two-step process, in which phosphorylation at the +2 position provides a negatively-charged specificity determinant that allows subsequent phosphorylation of Tyr184.

Amino Acid Isomerases↗
PubMed1997

Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: application of a differential interaction trap assay for examining protein-protein interactions.

Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organizes the components of the mitogen-activated protein kinase (MAPK) cascade. A new method was developed to isolate missense mutations in Ste5 that differentially affect the ability of Ste5 to interact with either of two MAPK cascade constituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affect association with Ste7 or with Ste11 delineate discrete regions of Ste5 that are critical for each interaction. Co-immunoprecipitation analysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G protein beta subunit), and Fus3 (MAPK), confirmed that each mutation specifically affects the interaction of Ste5 with only one protein. When expressed in a ste5 delta cell, mutant Ste5 proteins that are defective in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency. One mutation that clearly weakened (but did not eliminate) interaction of Ste5 with Ste7 permitted mating at wild-type efficiency, indicating that an efficacious signal is generated even when Ste5 associates with only a small fraction of (or only transiently with) Ste7. Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expressed, suggesting that the functional form of Ste5 in vivo is an oligomer.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Mck1, a member of the glycogen synthase kinase 3 family of protein kinases, is a negative regulator of pyruvate kinase in the yeast Saccharomyces cerevisiae.

An interaction between the Saccharomyces cerevisiae protein kinase Mck1 and pyruvate kinase (Pyk1) was detected by using the two-hybrid method. Purified Mck1 was able to phosphorylate purified Pyk1 on Ser in vitro. Pyruvate kinase activity was elevated in mck1 delta cells. Several of the phenotypes of mck1 delta mutants are similar to those observed in cells overexpressing PYK1. Co-overexpression of MCK1 suppressed all of the phenotypes associated with PYK1 overexpression. These results indicate that Mck1 negatively regulates pyruvate kinase activity, possibly by direct phosphorylation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Identification and characterization of the CLK1 gene product, a novel CaM kinase-like protein kinase from the yeast Saccharomyces cerevisiae.

The CLK1 gene of Saccharomyces cerevisiae encodes a 610-residue protein kinase that resembles known type II Ca2+/calmodulin-dependent protein kinases (CaM kinases), including the CMK1 and CMK2 gene products from the same yeast. The Clk1 kinase domain is preceded by a 162-residue N-terminal extension, followed by a 132-residue C-terminal extension (which contains a basic segment resembling known calmodulin-binding sites) and is as similar to mammalian CaM kinase (38% identity to rat CaM kinase alpha) as it is to yeast CaM kinase (37% identity to Cmk2). However, Clk1 shares 52% identity with Rck1, another putative protein kinase encoded in the S. cerevisiae genome. Clk1 tagged with a c-myc epitope (expressed in yeast) and a GST-Clk1 fusion (expressed in bacteria) underwent autophosphorylation and phosphorylated an exogenous substrate (yeast protein synthesis elongation factor 2), primarily on Ser. Neither Clk1 activity was stimulated by purified yeast calmodulin (CMD1 gene product), with or without Ca2+; no association of Clk1 with Cmd1 was detectable by other methods. C-terminally truncated Clk1(Delta487-610) was growth-inhibitory when overexpressed, whereas catalytically inactive Clk1(K201R Delta487-610) was not, suggesting that the C terminus is a negative regulatory domain. Using immunofluorescence, Clk1 was localized to the cytosol and excluded from the nucleus. A clk1Delta mutant, a clk1Delta rck1Delta double mutant, a clk1Delta cmk1Delta cmk2Delta triple mutant, and a clk1Delta rck1Delta cmk1Delta cmk2Delta quadruple mutant were all viable and manifested no other overt growth phenotype.

Amino Acid Sequence↗
PubMed1996

Two novel targets of the MAP kinase Kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae.

Haploid cells of budding yeast Saccharomyces cerevisiae respond to mating pheromones by inducing genes required for conjugation, arresting cell cycle progression, and undergoing morphological changes. The same cells respond to nutrient deprivation by altering budding pattern and inducing genes required for invasive growth. Both developmental alternatives to vegetative proliferation require the MAP kinase Kss1 and the transcriptional transactivator Ste12. Using a two-hybrid screen for gene products that interact with Kss1, two homologous and previously uncharacterized loci (DIG1 and DIG2, for down-regulator of invasive growth) were identified. DIG2 is pheromone-inducible, whereas DIG1 is constitutively expressed. Dig1 colocalizes with Kssl in the nucleus, coimmunoprecipitates with Kss1 from cell extracts in a pheromone-independent manner, and is phosphorylated by Kss1 in immune complexes in a pheromone-stimulated manner. Kss1 binds specifically to a GST-Dig1 fusion in the absence of any other yeast protein. Using the two-hybrid method, both Dig1 and Dig2 also interact with the other MAP kinase of the pheromone response pathway, Fus3. However, neither dig1 or dig2 single mutants, nor a dig1 dig2 double mutant, have a discernible effect on mating. In contrast, dig1 dig2 cells constitutively invade agar medium, whereas a dig1 dig2 ste12 triple mutant does not, indicating that Dig1 and Dig2 share a role in negatively regulating the invasive growth pathway. High-level expression of Dig1 suppresses invasive growth and also causes cells to appear more resistant to pheromone-imposed cell cycle arrest. Ste12 also binds specifically to GST-Dig1 in the absence of any other yeast protein. Collectively, these findings indicate that Dig1, and most likely Dig2, are physiological substrates of Kssl and suggest that they regulate Ste12 function by direct protein-protein interaction.

Amino Acid Sequence↗
PubMed1996

High-throughput screening for drug discovery.

Recent progress in elucidating the mechanisms underlying human disease has dramatically increased the number of protein targets available for potential drug treatment. Concurrently, new approaches have increased the number of compounds that can be tested for activity against these targets. Together, these trends have stimulated the adoption of high-throughput screening as a primary tool for early-stage drug discovery.

Drug Evaluation, Preclinical↗
PubMed1996

Immunophilins in the Yeast Saccharomyces cerevisiae: A Different Spin on Proline Rotamases

The clinically used immunosuppressant compounds-FK506, rapamycin, and cyclophilin A-are all natural products that were originally detected because of their antifungal action, not because of their fortuitous effects on the human immune system. Genetic and biochemical approaches have been used to identify binding proteins that serve as the receptors for these antibiotics in cells of the budding yeast Saccharomyces cerevisiae. Three FK506/rapamycin-binding proteins (FKBPs) and six cyclosporin-A-binding proteins (cyclophilins) have been characterized in some detail, but there is evidence that additional members of both families exist in this organism. Cloning of the corresponding genes has shown that the yeast gene products are strikingly similar to their mammalian counterparts and possess peptidylprolyl-cis, trans-isomerase (proline rotamase) activity in vitro. Genetic analysis in yeast has confirmed, and significantly extended, complementary research in animal cell systems that has shed light on the roles that the FKBPs and the cyclophilins play in the mechanism of action of the immunosuppressant drugs. The application of genetic methods in yeast is also beginning to provide additional insights into the function of these proteins in normal cell physiology.

Journal Article↗
PubMed1996

The PAL1 gene product is a peroxisomal ATP-binding cassette transporter in the yeast Saccharomyces cerevisiae.

The PAL1 gene was isolated using PCR and degenerate oligonucleotide primers corresponding to highly conserved amino acid sequence motifs diagnostic of the ATP-binding cassette domain of the superfamily of membrane-bound transport proteins typified by mammalian multidrug resistance transporter 1 and Saccharomyces cerevisiae Ste6. The deduced PAL1 gene product is similar in length to, has the same predicted topology as, and shares the highest degree of amino acid sequence identity with two human proteins, adrenoleukodystrophy protein and peroxisomal membrane protein (70 kD), which are both presumptive ATP-binding cassette transporters thought to be constituents of the peroxisomal membrane. As judged by hybridization of a PAL1 probe to isolated RNA and by expression of a PAL1-lacZ fusion, a PAL1 transcript was only detectable when cells were grown on oleic acid, a carbon source which requires the biogenesis of functional peroxisomes for its metabolism. A pal1delta mutant grew normally on either glucose- or glycerol-containing media; however, unlike PAL1+ cells (or the pal1delta mutant carrying the PAL1 gene on a plasmid), pal1delta cells were unable to grow on either a solid medium or a liquid medium containing oleic acid as the sole carbon source. Antibodies raised against a chimeric protein in which the COOH-terminal domain of Pal1 was fused to glutathione S-transferase specifically recognized a protein in extracts from wild-type cells only when grown on oleic acid; this species represents the PAL1 gene product because it was missing in pal1delta cells and more abundant in pal1delta cells expressing PAL1 from a multicopy plasmid. The Pal1 polypeptide was highly enriched in the organellar pellet fraction prepared from wild-type cells by differential centrifugation and comigrated upon velocity sedimentation in a Nycodenz gradient with a known component of the peroxisomal matrix, e-oxoacyl-CoA thiolase. As judged by both subcellular fractionation and indirect immunofluorescence, localization of 3-oxoacyl-CoA thiolase to peroxisomes was unchanged whether Pal1 was present, absent, or overexpressed. These findings demonstrate that Pal1 is a peroxisome-specific protein, that it is required for peroxisome function, but that it is not necessary for the biogenesis of peroxisomes or for the import of 3-oxoacyl-CoA thiolase (and at least two other peroxisomal matrix proteins).

ATP-Binding Cassette Transporters↗
PubMed1996

Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7.

Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), and Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone response pathway of Saccharomyces cerevisiae. To investigate the potential role of specific interactions between these enzymes during signaling, their ability to associate with each other was examined both in solution and in vivo. When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (Kd of approximately 5 nM) with Ste7 in the absence of any additional yeast proteins. These complexes were specific because neither Hog1 nor Mpk1 (two other yeast MAPKs), nor mammalian Erk2, was able to associate detectably with Ste7. Neither the kinase catalytic core of Ste7 nor the phosphoacceptor regions of Ste7 and Kss1 were necessary for complex formation. Ste7-Kss1 (and Ste7-Fus3) complexes were present in yeast cell extracts and were undiminished in extracts prepared from a ste5delta-ste11delta double mutant strain. In Ste7-Kss1 (or Ste7-Fus3) complexes isolated from naive or pheromone-treated cells, Ste7 phosphorylated Kss1 (or Fus3), and Kss1 (or Fus3) phosphorylated Ste7, in a pheromone-stimulated manner; dissociation of the high-affinity complex was shown to be required for either phosphorylation event. Deletions of Ste7 in the region required for its stable association with Kss1 and Fus3 in vitro significantly decreased (but did not eliminate) signaling in vivo. These findings suggest that the high-affinity and active site-independent binding observed in vitro facilitates signal transduction in vivo and suggest further that MEK-MAPK interactions may utilize a double-selection mechanism to ensure fidelity in signal transmission and to insulate one signaling pathway from another.

Base Sequence↗
PubMed1996

Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit).

Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST2 gene product fail to resume growth after exposure to pheromone. Conversely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detectably dampened signaling in a short-term assay of pheromone response (phosphorylation of Ste4, Gbeta subunit). When the GPA1 gene product (Galpha subunit) is absent, the pheromone response pathway is constitutively active and, consequently, growth ceases. Despite sustained induction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1delta mutants remain growth arrested, indicating that the action of Sst2 requires the presence of Gpa1. The N-terminal domain (residues 3 to 307) of Sst2 (698 residues) has sequence similarity to the catalytic regions of bovine GTPase-activating protein and human neurofibromatosis tumor suppressor protein; segments in the C-terminal domain of Sst2 (between residues 417 and 685) are homologous to other RGS proteins. Both the N- and C-terminal domains were required for Sst2 function in vivo. Consistent with a role for Sst2 in binding to and affecting the activity of Gpa1, the majority of Sst2 was membrane associated and colocalized with Gpa1 at the plasma membrane, as judged by sucrose density gradient fractionation. Moreover, from cell extracts, Sst2 could be isolated in a complex with Gpa1 (expressed as a glutathione S-transferase fusion); this association withstood the detergent and salt conditions required for extraction of these proteins from cell membranes. Also, SST2+ cells expressing a GTPase-defective GPA1 mutant displayed an increased sensitivity to pheromone, whereas sst2 cells did not. These results demonstrate that Sst2 and Gpa1 interact physically and suggest that Sst2 is a direct negative regulator of Gpa1.

Amino Acid Sequence↗
PubMed1995

The yeast immunophilin Fpr3 is a physiological substrate of the tyrosine-specific phosphoprotein phosphatase Ptp1.

The tyrosine-specific phosphoprotein phosphatase encoded by the Saccharomyces cerevisiae PTP1 gene dephosphorylates artificial substrates in vitro, but little is known about its functions and substrates in vivo. The presence of Ptp1 resulted in dephosphorylation of multiple tyrosine-phosphorylated proteins in yeast expressing a heterologous tyrosine-specific protein kinase, indicating that Ptp1 can dephosphorylate a broad range of substrates in vivo. Correspondingly, several proteins phosphorylated at tyrosine by endogenous protein kinases exhibited a marked increase in tyrosine phosphorylation in ptp1 mutant cells. One of these phosphotyrosyl proteins (p70) was also dephosphorylated in vitro when incubated with recombinant Ptp1. p70 was purified to homogeneity; analysis of four tryptic peptides revealed that p70 is identical to the recently described FPR3 gene product, a nucleolarly localized proline rotamase of the FK506- and rapamycin-binding family. The identity of p70 with Fpr3 was confirmed in the demonstration that the abundance of tyrosine-phosphorylated p70 in ptp1 mutants was strictly correlated with the level of FPR3 expression; immobilized phosphotyrosyl Fpr3 was directly dephosphorylated by recombinant Ptp1. Site-directed mutagenesis demonstrated that the site of tyrosine phosphorylation is Tyr-184, which resides within the nucleolin-like amino-terminal domain of Fpr3. Protein kinase activities from yeast cell extracts can bind to and phosphorylate the immobilized amino-terminal domain of Fpr3 on serine, threonine, and tyrosine. Fpr3 represents the first phosphotyrosyl protein identified in S. cerevisiae that is not itself a protein kinase and is as yet the only known physiological substrate of Ptp1.

Amino Acid Sequence↗
PubMed1995

Gain-of-function mutations in a human calmodulin-like protein identify residues critical for calmodulin action in yeast.

A human epithelial cell-specific transcript (NB-1) encodes a calmodulin-like protein (hCLP), which is identical in length and 85% identical in amino acid sequence to authentic human calmodulin (hCaM). Although hCaM shares only 60% amino acid sequence identity with yeast calmodulin (CMD1 gene product), hCaM was able to substitute functionally for Cmd1 in yeast cells. In contrast, hCLP was unable to support either spore germination or vegetative growth in Cmd1-deficient yeast cells, even when stably expressed at a level at least an order of magnitude above that of hCaM. Thus, hCLP provides an indicator protein for discerning those residues that are critical for calmodulin function in vivo. In addition to 20 conservative amino acid replacements, hCLP differs from hCaM (and other vertebrate calmodulins that are able to complement a cmd1 null mutation) by only three nonconservative substitutions. Site-directed mutagenesis was used to convert these three positions back to residues more typical of those found in authentic calmodulins and to prepare all possible combinations of these three mutations, specifically: three single mutants (R58V, R112N, and A128E), three double mutants (R58V A128E, R112N A128E, and R58V R112N), and the triple mutant (R58V R112N A128E). The triple mutant and one of the double mutants (R58V A128E) were able to restore an apparently normal growth rate to a cmd1 delta strain, indicating that the altered hCLPs have acquired the ability to behave as functional calmodulins in yeast. The other two double mutants were able to support growth of Cmd1-deficient cells only weakly, but cells expressing the R112N A128E mutant grew noticeably better than those expressing the R58V R112N mutant. Remarkably, one single mutant (A128E), but not the other two single mutants, was also reproducibly able to support weak growth of a cmd1 delta strain. The properties of these gain-of-function, or neomorphic, mutations implicate E128, and to a lesser extent V58, as residues critical for calmodulin action in vivo. Molecular modeling of these positions within the structure of a Ca(2+)-calmodulin.peptide complex indicates that E128 projects directly into the central cavity occupied by the bound peptide. Thus, E128 may contribute a contact that is vital for the interaction of Cmd1 with one or more of the targets that are essential for yeast cell growth.

Amino Acid Sequence↗
PubMed1995

Overexpression of the yeast MCK1 protein kinase suppresses conditional mutations in centromere-binding protein genes CBF2 and CBF5.

We find that overexpression in yeast of the yeast MCK1 gene, which encodes a meiosis and centromere regulatory kinase, suppresses the temperature-sensitive phenotype of certain mutations in essential centromere binding protein genes CBF2 and CBF5. Since Mck1p is a known serine/threonine protein kinase, this suppression is postulated to be due to Mck1p-catalyzed in vivo phosphorylation of centromere binding proteins. Evidence in support of this model was provided by the finding that purified Mck1p phosphorylates in vitro the 110 kDa subunit (Cbf2p) of the multimeric centromere binding factor CBF3. This phosphorylation occurs on both serine and threonine residues in Cbf2p.

Fungal Proteins↗
PubMed1995

Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway.

Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa haploids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo. De novo protein synthesis was required for sustained pheromone-induced phosphorylation of Kss1. Catalytically inactive Kss1 mutants displayed alpha-factor-induced phosphorylation on both residues, even in kss1 delta cells; hence, autophosphorylation is not obligatory for these modifications. In kss1 delta fus3 delta double mutants, Kss1 phosphorylation was elevated even in the absence of pheromone; thus, cross-phosphorylation by Fus3 is not responsible for Kss1 activation. In contrast, pheromone-induced Kss1 phosphorylation was eliminated in mutants deficient in two other protein kinases, Ste11 and Ste7. A dominant hyperactive allele of STE11 caused a dramatic increase in the phosphorylation of Kss1, even in the absence of pheromone stimulation, but required Ste7 for this effect, suggesting an order of function: Ste11-->Ste7-->Kss1. When overproduced, Kss1 stimulated recovery from pheromone-imposed G1 arrest. Catalytic activity was essential for Kss1 function in signal transmission, but not for its recovery-promoting activity. Kss1 was found almost exclusively in the particulate material and its subcellular fractionation was unaffected by pheromone treatment. Indirect immunofluorescence demonstrated that Kss1 is concentrated in the nucleus and that its distribution is not altered detectably during signaling.

Amino Acid Sequence↗
PubMed1994

Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism.

Basal transcription of many genes in yeast is repressed by Mot1, an essential protein which is a member of the Snf2/Swi2 family of conserved nuclear factors. ADI is an ATP-dependent inhibitor of TATA-binding protein (TBP) binding to DNA that inhibits transcription in vitro. Here we demonstrate that ADI is encoded by the MOT1 gene. Mutation of MOT1 abolishes ADI activity and derepresses basal transcription in vitro and in vivo. Recombinant Mot1 removes TBP from DNA and Mot1 contains an ATPase activity which is essential for its function. Genetic interactions between Mot1 and TBP indicate that their functions are interlinked in vivo. These results provide a general model for understanding the mechanism of action of a large family of nuclear factors involved in processes such as transcription and DNA repair.

Adenosine Triphosphatases↗