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M Tyers

Publications and source records attributed to M Tyers.

At least 19 recordsLinked to original sources
PubMed1998

Yeast G1 cyclins are unstable in G1 phase.

In most eukaryotes, commitment to cell division occurs in late G1 phase at an event called Start in the yeast Saccharomyces cerevisiae, and called the restriction point in mammalian cells. Start is triggered by the cyclin-dependent kinase Cdc28 and three rate-limiting activators, the G1 cyclins Cln1, Cln2 and Cln3. Cyclin accumulation in G1 is driven in part by the cell-cycle-regulated transcription of CLN1 and CLN2, which peaks at Start. CLN transcription is modulated by physiological signals that regulate G1 progression, but it is unclear whether Cln protein stability is cell-cycle-regulated. It has been suggested that once cells pass Start, Cln proteolysis is triggered by the mitotic cyclins Clb1, 2, 3 and 4. But here we show that G1 cyclins are unstable in G1 phase, and that Clb-Cdc28 activity is not needed fgr G1 cyclin turnover. Cln instability thus provides a means to couple Cln-Cdc28 activity to transcriptional regulation and protein synthetic rate in pre-Start G1 cells.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1998

Cdc53 is a scaffold protein for multiple Cdc34/Skp1/F-box proteincomplexes that regulate cell division and methionine biosynthesis in yeast.

In budding yeast, ubiquitination of the cyclin-dependent kinase (Cdk) inhibitor Sic1 is catalyzed by the E2 ubiquitin conjugating enzyme Cdc34 in conjunction with an E3 ubiquitin ligase complex composed of Skp1, Cdc53 and the F-box protein, Cdc4 (the SCFCdc4 complex). Skp1 binds a motif called the F-box and in turn F-box proteins appear to recruit specific substrates for ubiquitination. We find that Skp1 interacts with Cdc53 in vivo, and that Skp1 bridges Cdc53 to three different F-box proteins, Cdc4, Met30, and Grr1. Cdc53 contains independent binding sites for Cdc34 and Skp1 suggesting it functions as a scaffold protein within an E2/E3 core complex. F-box proteins show remarkable functional specificity in vivo: Cdc4 is specific for degradation of Sic1, Grr1 is specific for degradation of the G1 cyclin Cln2, and Met30 is specific for repression of methionine biosynthesis genes. In contrast, the Cdc34-Cdc53-Skp1 E2/E3 core complex is required for all three functions. Combinatorial control of SCF complexes may provide a basis for the regulation of diverse cellular processes.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1998

Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis.

The ubiquitin-dependent proteolytic pathway targets many key regulatory proteins for rapid intracellular degradation. Specificity in protein ubiquitination derives from E3 ubiquitin protein ligases, which recognize substrate proteins. Recently, analysis of the E3s that regulate cell division has revealed common themes in structure and function. One particularly versatile class of E3s, referred to as Skp1p-Cdc53p-F-box protein (SCF) complexes, utilizes substrate-specific adaptor subunits called F-box proteins to recruit various substrates to a core ubiquitination complex. A vast array of F-box proteins have been revealed by genome sequencing projects, and the early returns from genetic analysis in several organisms promise that F-box proteins will participate in the regulation of many processes, including cell division, transcription, signal transduction and development.

Animals↗
PubMed1997

F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex.

We have reconstituted the ubiquitination pathway for the Cdk inhibitor Sic1 using recombinant proteins. Skp1, Cdc53, and the F-box protein Cdc4 form a complex, SCFCdc4, which functions as a Sic1 ubiquitin-ligase (E3) in combination with the ubiquitin conjugating enzyme (E2) Cdc34 and E1. Cdc4 assembled with Skp1 functions as the receptor that selectively binds phosphorylated Sic1. Grr1, an F-box protein involved in Cln destruction, forms complexes with Skp1 and Cdc53 and binds phosphorylated Cln1 and Cln2, but not Sic1. Because the constituents of the SCF complex are members of protein families, SCFCdc4 is likely to serve as the prototype for a large class of E3s formed by combinatorial interactions of related family members. SCF complexes couple protein kinase signaling pathways to the control of protein abundance.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1997

Genomic organization and functional analysis of the murine protein phosphatase 1c gamma (Ppp1cc) gene.

Protein phosphatase 1 holoenzymes are composed of catalytic subunits in combination with various regulatory subunits. In rodents, four different catalytic isoforms are known, PP1c alpha, -delta, -gamma 1, and -gamma 2. Here we describe the genomic organization of the murine Ppp1cc gene that encodes the PP1c gamma 1 and PP1c gamma 2 isoforms. We determined that Ppp1cc maps to F1.2-G1.2 on chromosome 5 by FISH mapping. Southern hybridization and analysis of cross-hybridizing genomic clones revealed four Ppp1cc-related pseudogenes in the mouse genome. The authentic Ppp1cc gene encodes two isoforms, PP1c gamma 1 and PP1c gamma 2, that arise from alternative splicing and differ by retention of the last intron. The introns of Ppp1cc are flanked by short direct repeats, the significance of which is not clear. Both isoforms retain phosphatase function since they are able to complement the cold-sensitive PP1 defect caused by the dis2-11 mutation in the fission yeast Schizosaccharomyces pombe.

Amino Acid Sequence↗
PubMed1997

Deregulation of CLN1 and CLN2 in the Saccharomyces cerevisiae whi2 mutant.

Wild-type cells of the budding yeast Saccharbmyces cerevisiae arrest in G1 upon nutrient exhaustion. Cell cycle arrest requires the WHI2 gene since whi2 mutants continue to divide and become abnormally small as nutrients are depleted. Here we show that CLN1 and CLN2 transcript levels in a whi2 strain are higher during exponential growth, and persist longer upon starvation, than in an isogenic wild-type strain. In contrast to CLN1 and CLN2, CLN3 levels declined only at very high cell density and were unaffected by the whi2 mutation. Elevated CLN expression is sufficient to explain the whi2 phenotype since ectopic expression of CLN1 in a nutrient-depleted culture caused cells to continue dividing and interfered with the acquisition of heat resistance. These observations show that, either directly or indirectly, Whi2 negatively regulates G1 cyclin expression. Interestingly extremely high levels of Cln1 induced filamentous growth upon nutrient deprivation, suggesting a direct connection between G1 cyclin activity and morphological responses to poor nutrient conditions.

Cyclins↗
PubMed1997

Phosphorylation and subcellular redistribution of pleckstrin in human neutrophils.

Pleckstrin, originally described as a major substrate of protein kinase C (PKC) in platelets, was found to be highly expressed in human neutrophils (intracellular concentration, approximately 15 microM). As PKC isoforms play an important role in mediating neutrophil antimicrobial responses, we studied the regulation of pleckstrin phosphorylation in response to inflammatory stimuli. Following treatment of neutrophils with FMLP, 12-O-tetradecanoylphorbol-13-acetate, or opsonized zymosan, pleckstrin was rapidly phosphorylated, which resulted in a shift in its electrophoretic mobility. Several lines of evidence suggest that pleckstrin is phosphorylated in part by a nonconventional PKC following stimulation by FMLP: 1) chelation of intracellular Ca2+ had only a partial inhibitory effect; 2) diacylglycerol kinase inhibitors shortened the duration of phosphorylation, while the phosphatidic acid phosphohydrolase antagonist propranolol extended it; and 3) wortmannin and erbstatin blocked the phosphorylation of pleckstrin. These results suggest that nonconventional PKC isoforms, possibly delta or zeta, mediate the phosphorylation of pleckstrin. Both PKCdelta and -zeta are expressed in human neutrophils. Increased association of pleckstrin with both microsomes and with the cytoskeleton was observed in stimulated cells. These findings suggest that phosphorylation by nonconventional PKC isoforms induces a conformational change in pleckstrin that promotes its interaction with membranes and/or with the cytoskeleton. Such a translocation may serve to target proteins or lipids recognized by pleckstrin homology domains to sites where they can contribute to the microbicidal response.

Androstadienes↗
PubMed1997

Regulation of the mating pheromone and invasive growth responses in yeast by two MAP kinase substrates.

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, components of a single mitogen-activated protein (MAP) kinase pathway transduce two distinct signals, each of which activates an independent developmental programme: peptide mating pheromones initiate the mating response, whereas nutrient limitation initiates filamentous growth. One of the MAP kinases in this pathway, Fus3, triggers mating but antagonizes filamentous growth, while the other, Kss 1, preferentially triggers filamentous growth. Both kinases activate the same transcription factor, Ste 12, which can stimulate gene expression specific to each of the developmental programmes. The precise mechanism by which these MAP kinases activate Ste 12, however, is not clear. RESULTS: Two newly identified proteins, Rst 1 and Rst 2 (also known as Dig1 and Dig2), were found to associate physically with Fus3 and Ste12. Rst1 and Rst2 were prominent substrates in kinase reactions of Fus3 immune complexes from pheromone-treated cells. Association of Fus3 with Ste12 required Rst1 and Rst2, and activation of Fus3 by pheromone caused release of Ste12 from the Fus3 complex. Although rst1 and rst2 single mutants had no obvious phenotype, both filamentous growth and mating-specific gene expression were constitutive in rst1 rst2 double mutants. The phenotype of rst1 rst2 cells required Ste12 function, but did not require the function of upstream kinases. Consistent with Rst1 and Rst2 having a role in Ste12 regulation, both proteins were localized to the nucleus. CONCLUSIONS: Rst1 and Rst2 repress the mating and filamentous growth responses of S. cerevisiae by directly inhibiting Ste12. Activation of Fus3 or Kss1 may cause phosphorylation-dependent release of Ste12 from Rst1/Rst2 and thereby activate Ste12-dependent transcription.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Human CPR (cell cycle progression restoration) genes impart a Far- phenotype on yeast cells.

Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexpressed were capable of specifically overcoming G1 arrest signals from the cell cycle branch of the mating pheromone pathway, while still maintaining the integrity of the transcriptional induction branch. We have identified 13 human CPR (cell cycle progression restoration) genes and 11 yeast OPY (overproduction-induced pheromone-resistant yeast) genes that specifically block the G1 arrest by mating pheromone. The CPR genes represent a variety of biochemical functions including a new cyclin, a tumor suppressor binding protein, chaperones, transcription factors, translation factors, RNA-binding proteins, as well as novel proteins. Several CPR genes require individual CLNs to promote pheromone resistance and those that require CLN3 increase the basal levels of Cln3 protein. Moreover, several of the yeast OPY genes have overlapping functions with the human CPR genes, indicating a possible conservation of roles.

Amino Acid Sequence↗
PubMed1996

Cdc53 targets phosphorylated G1 cyclins for degradation by the ubiquitin proteolytic pathway.

In budding yeast, cell division is initiated in late G1 phase once the Cdc28 cyclin-dependent kinase is activated by the G1 cyclins Cln1, Cln2, and Cln3. The extreme instability of the Cln proteins couples environmental signals, which regulate Cln synthesis, to cell division. We isolated Cdc53 as a Cln2-associated protein and show that Cdc53 is required for Cln2 instability and ubiquitination in vivo. The Cln2-Cdc53 interaction, Cln2 ubiquitination, and Cln2 instability all depend on phosphorylation of Cln2. Cdc53 also binds the E2 ubiquitin-conjugating enzyme, Cdc34. These findings suggest that Cdc53 is a component of a ubiquitin-protein ligase complex that targets phosphorylated G1 cyclins for degradation by the ubiquitin-proteasome pathway.

Amino Acid Sequence↗
PubMed1996

The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirement for Cln G1 cyclin function at Start.

In yeast, commitment to cell division (Start) is catalyzed by activation of the Cdc28 protein kinase in late G1 phase by the Cln1, Cln2, and Cln3 G1 cyclins. The Clns are essential, rate-limiting activators of Start because cells lacking Cln function (referred to as cln-) arrest at Start and because CLN dosage modulates the timing of Start. At or shortly after Start, the development of B-type cyclin Clb-Cdc28 kinase activity and initiation of DNA replication requires the destruction of p40SIC1, a specific inhibitor of the Clb-Cdc28 kinases. I report here that cln cells are rendered viable by deletion of SIC1. Conversely, in cln1 cln2 cells, which have low CLN activity, modest increases in SIC1 gene dosage cause inviability. Deletion of SIC1 does not cause a general bypass of Start since (cln-)sic1 cells remain sensitive to mating pheromone-induced arrest. Far1, a pheromone-activated inhibitor of Cln-Cdc28 kinases, is dispensable for arrest of (cln-)sic1 cells by pheromone, implying the existence of an alternate Far1-independent arrest pathway. These observations define a pheromone-sensitive activity able to catalyze Start only in the absence of p40SIC1. The existence of this activity means that the B-type cyclin inhibitor p40SIC1 imposes the requirement for Cln function at Start.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1996

A new family of regulators of G-protein-coupled receptors?

Organisms as diverse as fungi and humans use G-protein-coupled receptors to control signal transduction pathways responsive to various hormones, neuroregulatory molecules and other sensory stimuli. Continual stimulation of these receptors often leads to their desensitization, which is mediated in part by the consecutive actions of two families of proteins--the G-protein-coupled receptor kinases, which phosphorylate the agonist-occupied receptors, and the arrestin proteins, which subsequently bind to the receptors. We now present evidence that a group of proteins--the G0S8/Sst2p family--may be a third class of receptor-desensitizing factors.

Amino Acid Sequence↗
PubMed1994

The PCL2 (ORFD)-PHO85 cyclin-dependent kinase complex: a cell cycle regulator in yeast.

Cyclin-dependent kinase (cdk) complexes are essential activators of cell cycle progression in all eukaryotes. In contrast to mammalian cells, in which multiple cdk's contribute to cell cycle regulation, the yeast cell cycle is largely controlled by the activity of a single cdk, CDC28. Analysis of the putative G1 cyclin PCL2 (ORFD) identified a second cyclin-cdk complex that contributes to cell cycle progression in yeast. PCL2 interacted with the cdk PHO85 in vivo and in vitro and formed a kinase complex that had G1-periodic activity. Under genetic conditions in which the Start transition was compromised, PHO85 and its associated cyclin subunits were essential for cell cycle commitment. Because PHO85 and another cyclin-like molecule, PHO80, also take part in inorganic phosphate metabolism, this cdk enzyme may integrate responses to nutritional conditions with the cell cycle.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1994

Inhibition of G1 cyclin activity by the Ras/cAMP pathway in yeast.

In the yeast Saccharomyces cerevisiae, commitment to cell division (Start) requires growth to a critical cell size. The G1 cyclins Cln1, Cln2 and Cln3 activate the Cdc28 protein kinase and are rate-limiting activators of Start. When glucose is added to cells growing in a poor carbon source, the critical cell size required for Start is reset from a small to a large size. In yeast, glucose acts through Ras proteins to stimulate adenylyl cyclase, activating the three cyclic AMP-dependent protein kinases Tpk1, Tpk2 and Tpk3 (refs 8, 9). We find that stimulation of the Ras/cAMP pathway represses expression of CLN1, CLN2 and co-regulated genes, inhibiting Start. This helps explain the increase in critical size when cells are shifted from poor to rich medium. This connection between the molecules controlling growth (Ras/cAMP) and those controlling division (cyclins) helps explain how division is co-ordinated with growth.

Cell Division↗
PubMed1993

Mechanisms that help the yeast cell cycle clock tick: G2 cyclins transcriptionally activate G2 cyclins and repress G1 cyclins.

In budding yeast, G1 cyclins such as CLN1 and CLN2 are expressed in G1 and S phases, while mitotic cyclins such as CLB1 and CLB2 are expressed in G2 and M phases. We find that the CLBs play a central role in the transition from CLNs to CLBs: the CLBs stimulate their own expression while repressing that of CLNs. This negative regulation of CLNs may occur via the transcription factor SWI4, because CLBs are necessary for G2 repression of SCB-regulated genes like CLN1 and CLN2 but not for repression of MCB-regulated genes like DNA polymerase and CLB5. Furthermore, SW14 associates with CLB2 protein and is a substrate for the CLB2-associated CDC28 kinase in vitro.

Activity Cycles↗
PubMed1993

Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins.

In the budding yeast Saccharomyces cerevisiae, the G1 cyclins Cln1, Cln2 and Cln3 regulate entry into the cell cycle (Start) by activating the Cdc28 protein kinase. We find that Cln3 is a much rarer protein than Cln1 or Cln2 and has a much weaker associated histone H1 kinase activity. Unlike Cln1 and Cln2, Cln3 is not significantly cell cycle regulated, nor is it down-regulated by mating pheromone-induced G1 arrest. An artificial burst of CLN3 expression early in G1 phase accelerates Start and rapidly induces at least five other cyclin genes (CLN1, CLN2, HCS26, ORFD and CLB5) and the cell cycle-specific transcription factor SWI4. In similar experiments, CLN1 is less efficient than CLN3 at activating Start. Strikingly, expression of HCS26, ORFD and CLB5 is dependent on CLN3 in a cln1 cln2 strain, possibly explaining why CLN3 is essential in the absence of CLN1 and CLN2. To explain the potent ability of Cln3 to activate Start, despite its apparently weak biochemical activity, we propose that Cln3 may be an upstream activator of the G1 cyclins which directly catalyze Start. Given the large number of known cyclins, such cyclin cascades may be a common theme in cell cycle control.

Base Sequence↗
PubMed1993

Far1 and Fus3 link the mating pheromone signal transduction pathway to three G1-phase Cdc28 kinase complexes.

In the yeast Saccharomyces cerevisiae, the Cdc28 protein kinase controls commitment to cell division at Start, but no biologically relevant G1-phase substrates have been identified. We have studied the kinase complexes formed between Cdc28 and each of the G1 cyclins Cln1, Cln2, and Cln3. Each complex has a specific array of coprecipitated in vitro substrates. We identify one of these as Far1, a protein required for pheromone-induced arrest at Start. Treatment with alpha-factor induces a preferential association and/or phosphorylation of Far1 by the Cln1, Cln2, and Cln3 kinase complexes. This induced interaction depends upon the Fus3 protein kinase, a mitogen-activated protein kinase homolog that functions near the bottom of the alpha-factor signal transduction pathway. Thus, we trace a path through which a mitogen-activated protein kinase regulates a Cdc2 kinase.

Amino Acid Sequence↗