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1.
Mol Cell ; 68(6): 1120-1133.e3, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29249654

RESUMO

The ability to respond to available nutrients is critical for all living cells. The AMP-activated protein kinase (SNF1 in yeast) is a central regulator of metabolism that is activated when energy is depleted. We found that SNF1 activity in the nucleus is regulated by controlled relocalization of the SNF1 activator Std1 into puncta. This process is regulated by glucose through the activity of the previously uncharacterized protein kinase Vhs1 and its substrate Sip5, a protein of hitherto unknown function. Phosphorylation of Sip5 prevents its association with Std1 and triggers Std1 accretion. Reversible Std1 puncta formation occurs under non-stressful, ambient conditions, creating non-amyloid inclusion bodies at the nuclear-vacuolar junction, and it utilizes cellular chaperones similarly to the aggregation of toxic or misfolded proteins such as those associated with Parkinson's, Alzheimer's, and CJD diseases. Our results reveal a controlled, non-pathological, physiological role of protein aggregation in the regulation of a major metabolic cellular pathway.


Assuntos
Glucose/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Agregados Proteicos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Redes e Vias Metabólicas , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Edulcorantes/farmacologia
2.
Int J Mol Sci ; 24(2)2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36674878

RESUMO

Energy status in all eukaryotic cells is sensed by AMP-kinases. We have previously found that the poly-histidine tract at the N-terminus of S. cerevisiae AMPK (Snf1) inhibits its function in the presence of glucose via a pH-regulated mechanism. We show here that in the absence of glucose, the poly-histidine tract has a second function, linking together carbon and iron metabolism. Under conditions of iron deprivation, when different iron-intense cellular systems compete for this scarce resource, Snf1 is inhibited. The inhibition is via an interaction of the poly-histidine tract with the low-iron transcription factor Aft1. Aft1 inhibition of Snf1 occurs in the nucleus at the nuclear membrane, and only inhibits nuclear Snf1, without affecting cytosolic Snf1 activities. Thus, the temporal and spatial regulation of Snf1 activity enables a differential response to iron depending upon the type of carbon source. The linkage of nuclear Snf1 activity to iron sufficiency ensures that sufficient clusters are available to support respiratory enzymatic activity and tests mitochondrial competency prior to activation of nuclear Snf1.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Fosforilação , Proteínas de Saccharomyces cerevisiae/metabolismo , Carbono/metabolismo , Ferro/metabolismo , Glucose/metabolismo
3.
EMBO J ; 36(4): 425-440, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28069708

RESUMO

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Humanos , Proteínas dos Microfilamentos/química , Ubiquitina-Proteína Ligases Nedd4 , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Complexo de Endopeptidases do Proteassoma/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Proteínas de Saccharomyces cerevisiae/química
4.
Curr Genet ; 65(6): 1321-1323, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31119370

RESUMO

Most organisms prefer to utilize glucose as a carbon source. Accordingly, the expression of genes involved in the catabolism of other carbon sources is repressed by the presence of glucose in a process known as (carbon) catabolite repression. However, much less is known about the relationships between "poor" carbon sources. We have recently shown that the enzyme alcohol dehydrogenase of the yeast Saccharomyces cerevisiae (ADH2), required for the utilization of ethanol, is not only inhibited by glucose, but by the acetate imported from the medium or produced by ethanol metabolism. Our study showed that sensing of acetate takes place within the cell, and not in the external medium, and that "poor" carbon sources are also utilized according to a pre-established hierarchy.


Assuntos
Ácido Acético/metabolismo , Repressão Catabólica , Glucose/metabolismo , Saccharomyces cerevisiae/metabolismo , Álcool Desidrogenase/metabolismo , Etanol/metabolismo , Regulação Fúngica da Expressão Gênica , Glicerol/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Curr Genet ; 64(4): 785-788, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29322248

RESUMO

Glucose is the preferred carbon of the yeast Saccharomyces cerevisiae. Depletion of glucose activates SNF1 (yeast AMP-activated protein kinase-AMPK), allowing cells to switch from fermentation to respiration. We have recently characterized the mechanism by which SNF1 activity is regulated by the Std1 protein, and its regulator Sip5. The hitherto uncharacterized protein kinase Vhs1 phosphorylates Sip5 in response to glucose availability, disengaging it from Std1 and promoting the sequestering of the SNF1 activator out of the nucleus into cytoplasmic puncta. These aggregates, which have the properties of liquid drops, and not of amyloids, reside in the nucleus-vacuole junction. The process is reversible, and Std1 puncta dissolve when glucose becomes scarce again. This reversible process requires protein chaperones, similar to the aggregation of toxic or misfolded proteins such as those associated with Huntington's Chorea, Alzheimer's and CJD diseases. Our results thus reveal a regulated, non-pathological, physiological role of protein aggregation that controls a major metabolic cellular pathway.


Assuntos
Glucose/metabolismo , Redes e Vias Metabólicas/genética , Agregados Proteicos/genética , Saccharomyces cerevisiae/genética , Núcleo Celular/genética , Fermentação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/genética
6.
Proc Natl Acad Sci U S A ; 110(43): 17432-7, 2013 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-24108357

RESUMO

The AMP-activated protein kinase (AMPK) is a major stress sensor of mammalian cells. AMPK's homolog in the yeast Saccharomyces cerevisiae, the SNF1 protein kinase, is a central regulator of carbon metabolism that inhibits the Snf3/Rgt2-Rgt1 glucose sensing pathway and activates genes involved in respiration. We present evidence that glucose induces modification of the Snf1 catalytic subunt of SNF1 with the small ubiquitin-like modifier protein SUMO, catalyzed by the SUMO (E3) ligase Mms21. Our results suggest that SUMOylation of Snf1 inhibits its function in two ways: by interaction of SUMO attached to lysine 549 with a SUMO-interacting sequence motif located near the active site of Snf1, and by targeting Snf1 for destruction via the Slx5-Slx8 (SUMO-directed) ubiquitin ligase. These findings reveal another way SNF1 function is regulated in response to carbon source.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucose/farmacologia , Immunoblotting , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Estabilidade Proteica , Proteína SUMO-1/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sumoilação
7.
Biology (Basel) ; 12(7)2023 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-37508436

RESUMO

Snf1, the fungal homologue of mammalian AMP-dependent kinase (AMPK), is a key protein kinase coordinating the response of cells to a shortage of glucose. In fungi, the response is to activate respiratory gene expression and metabolism. The major regulation of Snf1 activity has been extensively investigated: In the absence of glucose, it becomes activated by phosphorylation of its threonine at position 210. This modification can be erased by phosphatases when glucose is restored. In the past decade, two additional independent mechanisms of Snf1 regulation have been elucidated. In response to glucose (or, surprisingly, also to DNA damage), Snf1 is SUMOylated by Mms21 at lysine 549. This inactivates Snf1 and leads to Snf1 degradation. More recently, glucose-induced proton export has been found to result in Snf1 inhibition via a polyhistidine tract (13 consecutive histidine residues) at the N-terminus of the Snf1 protein. Interestingly, the polyhistidine tract plays also a central role in the response to iron scarcity. This review will present some of the glucose-sensing mechanisms of S. cerevisiae, how they interact, and how their interplay results in Snf1 inhibition by three different, and independent, mechanisms.

8.
iScience ; 25(10): 105083, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36147951

RESUMO

Cellular regulation of pH is crucial for internal biological processes and for the import and export of ions and nutrients. In the yeast Saccharomyces cerevisiae, the major proton pump (Pma1) is regulated by glucose. Glucose is also an inhibitor of the energy sensor Snf1/AMPK, which is conserved in all eukaryotes. Here, we demonstrate that a poly-histidine (polyHIS) tract in the pre-kinase region (PKR) of Snf1 functions as a pH-sensing module (PSM) and regulates Snf1 activity. This regulation is independent from, and unaffected by, phosphorylation at T210, the major regulatory control of Snf1, but is controlled by the Pma1 plasma-membrane proton pump. By examining the PKR from additional yeast species, and by varying the number of histidines in the PKR, we determined that the polyHIS functions progressively. This regulation mechanism links the activity of a key enzyme with the metabolic status of the cell at any given moment.

9.
Curr Opin Cell Biol ; 69: 1-6, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33388622

RESUMO

Many cellular processes involve buffering mechanisms against noise to enhance state stability. Such processes include the cell cycle and the switch between respiration and fermentation. In recent years, protein aggregation/condensation has emerged as an important regulatory mechanism. In this article, we examine the regulation of Std1, an activator of the Snf1/AMPK kinase, by sequestration into foci of liquid drops, and how foci of metabolic signaling and enzymatic proteins are regulated by chaperones, anti-aggregases and by phosphorylation.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Glucose , Fosforilação , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
10.
J Mol Biol ; 433(23): 167276, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34599943

RESUMO

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.


Assuntos
Bioensaio/métodos , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Expressão Gênica , Genes Reporter , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise
11.
Biochem Soc Trans ; 38(Pt 1): 78-82, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20074039

RESUMO

The APC/C (anaphase-promoting complex/cyclosome) discovered exactly 15 years ago by Avram Heshko and Marc Kirschner is by far the most complex ubiquitin ligase discovered so far. The APC/C is composed of roughly a dozen subunits and measures a massive 1.5 MDa. This huge complex, as well as its multiple modes of regulation, boasts impressive evolutionary conservation. One of its most puzzling features is its split personality: regulation of mitotic exit events on the one hand, and its ongoing activity during G(1)-phase, G(0)-phase and in terminally differentiated cells. The present short review is intended to provide a basic description of our current understanding of the APC/C, focusing on recent findings concerning its role in G(1)-phase and in differentiated cells.


Assuntos
Complexos Ubiquitina-Proteína Ligase/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Ciclo Celular/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genética
12.
Sci Rep ; 9(1): 6491, 2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-31019232

RESUMO

Cells adapt their gene expression and their metabolism in response to a changing environment. Glucose represses expression of genes involved in the catabolism of other carbon sources in a process known as (carbon) catabolite repression. However, the relationships between "poor" carbon sources is less characterized. Here we show that in addition to the well-characterized glucose (and galactose) repression of ADH2 (alcohol dehydrogenase 2, required for efficient utilization of ethanol as a carbon source), ADH2 expression is also inhibited by acetate which is produced during ethanol catabolism. Thus, repressive regulation of gene expression occurs also between "poor" carbon sources. Acetate repression of ADH2 expression is via Haa1, independently from the well-characterized mechanism of AMPK (Snf1) activation of Adr1. The response to extracellular acetate is attenuated when all three acetate transporters (Ady2, Fps1 and Jen1) are deleted, but these deletions do not affect the acetate response resulting from growth with glucose or ethanol as the carbon source. Furthermore, genetic manipulation of the ethanol catabolic pathway affects this response. Together, our results show that acetate is sensed intracellularly and that a hierarchical control of carbon sources exists even for "poor" carbon sources.


Assuntos
Carbono/metabolismo , Repressão Catabólica , Glucose/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetatos/metabolismo , Acetatos/farmacologia , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Etanol/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Methods Mol Biol ; 1342: 321-36, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26254934

RESUMO

The eukaryotic cell cycle is comprised of different phases that take place sequentially once, and normally only once, every division cycle. Such a dynamic process is best viewed in real time in living dividing cells. The insights that can be gained from such methods are considerably larger than any alternative technique that only generates snapshots. A great number of studies can gain from live cell imaging; however this method often feels somewhat intimidating to the novice. The purpose of this chapter is to demonstrate that imaging cell cycle phases in living cells from yeast to human is relatively easy and can be performed with equipment that is available in most research institutes. We present the different approaches, review different types of reporters, and discuss in depth all the aspects to be considered to obtain optimal results. We also describe our latest cell cycle markers, which afford unprecedented "sub"-phase temporal resolution.


Assuntos
Ciclo Celular , Imagem Molecular/métodos , Saccharomycetales/citologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Camundongos , Células NIH 3T3
14.
Cell Cycle ; 14(19): 3138-45, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26252546

RESUMO

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C(Cdh1) mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1(m11) mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C(Cdh1) substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Proteínas Cdh1/genética , Proteínas Cdh1/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Ciclina B/genética , Ciclina B/metabolismo , Replicação do DNA/genética , Replicação do DNA/fisiologia , Mitose/genética , Mitose/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética
15.
Cell Rep ; 12(11): 1865-75, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26344768

RESUMO

Yeast cells with DNA damage avoid respiration, presumably because products of oxidative metabolism can be harmful to DNA. We show that DNA damage inhibits the activity of the Snf1 (AMP-activated) protein kinase (AMPK), which activates expression of genes required for respiration. Glucose and DNA damage upregulate SUMOylation of Snf1, catalyzed by the SUMO E3 ligase Mms21, which inhibits SNF1 activity. The DNA damage checkpoint kinases Mec1/ATR and Tel1/ATM, as well as the nutrient-sensing protein kinase A (PKA), regulate Mms21 activity toward Snf1. Mec1 and Tel1 are required for two SNF1-regulated processes-glucose sensing and ADH2 gene expression-even without exogenous genotoxic stress. Our results imply that inhibition of Snf1 by SUMOylation is a mechanism by which cells lower their respiration in response to DNA damage. This raises the possibility that activation of DNA damage checkpoint mechanisms could contribute to aerobic fermentation (Warburg effect), a hallmark of cancer cells.


Assuntos
Dano ao DNA , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação
16.
Nat Commun ; 6: 7075, 2015 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-25959309

RESUMO

Ndd1 activates the Mcm1-Fkh2 transcription factor to transcribe mitotic regulators. The anaphase-promoting complex/cyclosome activated by Cdh1 (APC/C(Cdh1)) mediates the degradation of proteins throughout G1. Here we show that the APC/C(Cdh1) ubiquitinates Ndd1 and mediates its degradation, and that APC/C(Cdh1) activity suppresses accumulation of Ndd1 targets. We confirm putative Ndd1 targets and identify novel ones, many of them APC/C(Cdh1) substrates. The APC/C(Cdh1) thus regulates these proteins in a dual manner­both pretranscriptionally and post-translationally, forming a multi-layered feedforward loop (FFL). We predict by mathematical modelling and verify experimentally that this FFL introduces a lag between APC/C(Cdh1) inactivation at the end of G1 and accumulation of genes transcribed by Ndd1 in G2. This regulation generates two classes of APC/C(Cdh1) substrates, early ones that accumulate in S and late ones that accumulate in G2. Our results show how the dual state APC/C(Cdh1) activity is converted into multiple outputs by interactions between its substrates.


Assuntos
Proteínas Cdh1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Mitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Cdh1/genética , Proteínas de Ciclo Celular/genética , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
17.
Cell Div ; 6: 23, 2011 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-22204387

RESUMO

BACKGROUND: Cdc5 (polo kinase/Plk1) is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28) phosphorylation site present. FINDINGS: Cdc5T23A introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts in cis and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/CCdh1 independent and requires new protein synthesis. Cdc5T23E is hyperactive, and reduces the levels of Cdc5 (in trans) and drastically reduces Clb2 levels. CONCLUSIONS: Phosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1) caps Cdc5 and other CLB2 cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/CCdh1 inactivation.

18.
PLoS One ; 5(12): e15089, 2010 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-21151918

RESUMO

The Swe1/Wee1 kinase phosphorylates and inhibits Cdk1-Clb2 and is a major mitotic switch. Swe1 levels are controlled by ubiquitin mediated degradation, which is regulated by interactions with various mitotic kinases. We have recently reported that Swe1 levels are capable of sensing the progress of the cell cycle by measuring the levels of Cdk1-Clb2, Cdc5 and Hsl1. We report here a novel mechanism that regulates the levels of Swe1. We show that S. cerevisiae Swe1 is modified by Smt3/SUMO on residue K594 in a Cdk1 dependant manner. A degradation of the swe1(K594R) mutant that cannot be modified by Smt3 is considerably delayed in comparison to wild type Swe1. Swe1(K594R) cells express elevated levels of Swe1 protein and demonstrate higher levels of Swe1 activity as manifested by Cdk1-Y19 phosphorylation. Interestingly this mutant is not targeted, like wild type Swe1, to the bud neck where Swe1 degradation takes place. We show that Swe1 is SUMOylated by the Siz1 SUMO ligase, and consequently siz1Δ cells express elevated levels of Swe1 protein and activity. Finally we show that swe1(K594R) cells are sensitive to osmotic stress, which is in line with their compromised regulation of Swe1 degradation.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Tirosina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sequência de Aminoácidos , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/química , Mitose , Dados de Sequência Molecular , Mutação , Osmose , Fosforilação , Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismo
19.
Cell Cycle ; 9(15): 3046-53, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20714223

RESUMO

Swe1/Wee1 regulates mitotic entry by inhibiting Clb2-Cdk1 and its accumulation is involved in stress induced G(2) arrest. The APC/C(Cdh1) substrates Cdc5, Clb2 and Hsl1 regulate Swe1 degradation. We observed that clb2Deltacdh1Delta double mutant S. cerevisiae does not express any detectable levels of Swe1, presumably due to its constitutive degradation. This effect of Cdh1 inactivation is due to stabilization of Cdc5 and Hsl1, as expression of the non-degradable Cdc5(T29A) in clb2Delta cells prevented Swe1 accumulation. Strikingly, expression of non-degradable Hsl1(mdb/mkb) prevented Swe1 accumulation even in wild type Clb2 cells. Interestingly Swe1 accumulation could be reconstituted in all these mutants by eliciting a replication fork stress with hydroxyurea. Cells expressing the Clb2(ME) mutant, that cannot bind Swe1, behaved like clb2Delta cells, and failed to accumulate Swe1 in the absence of Cdh1 or the presence of Cdc5(T29A). This suggests that for Swe1 to accumulate it must interact with Clb2. We further show that in the absence of Clb2, Hsl1 is no longer essential for Swe1 degradation. We hypothesize that Clb2-Cdk1 protects Swe1 from premature degradation until its Hsl1 mediated de-protection, which enables its Cdc5 mediated degradation. Swe1 levels are thus regulated by monitoring the levels of three major mitotic regulators.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Estabilidade Enzimática , Modelos Biológicos , Mutação/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/citologia , Estresse Fisiológico
20.
Cell Cycle ; 8(18): 3003-9, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19713762

RESUMO

Cdh1 activates the Anaphase Promoting Complex/Cyclosome (APC/C(Cdh1)) throughout G(1) to degrade key cell cycle proteins. Cdh1 is not essential for cell proliferation, in spite of the fact that overexpression of some its degradation substrates is highly toxic. We report here that cdh1Delta cells are sensitive to stresses that activate the CWI (Cell Wall Integrity) and Hog1 MAP kinase pathways. Stresses did not activate APC/C(Cdh1) and cellular sensitivity was thus clearly due to constitutively elevated substrate levels. To explore the contribution of stabilization of individual APC/C(Cdh1) substrates to stress sensitivity, we generated cell lines expressing stabilized substrate mutants under their endogenous promoters. Cells expressing stabilized Hsl1 were sensitive to caffeine and failed to activate the Slt2 pathway. Cells expressing partially stable Clb2 were particularly sensitive to different stresses, possibly due to reduced Sic1 levels. Cells expressing stabilized Cdc5 were much less stress sensitive. Interestingly sensitivity of cdh1Delta cells does not seem to be restricted to G(1) but is manifested also during S and G(2) when the APC/C(Cdh1) is inactive anyway. We thus hypothesize that a role of G(1) specific APC/C(Cdh1) activity is to reset substrate levels to enables appropriate regulation of substrate accumulation in the subsequent phases of the cell cycle.


Assuntos
Adaptação Fisiológica , Ciclina B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Complexos Ubiquitina-Proteína Ligase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdh1 , Ciclo Celular , Fase G1 , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estabilidade Proteica , Saccharomyces cerevisiae/citologia , Estresse Fisiológico
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