scholarly journals Phosphorylation of the Mitotic Regulator Protein Hec1 by Nek2 Kinase Is Essential for Faithful Chromosome Segregation

2002 ◽  
Vol 277 (51) ◽  
pp. 49408-49416 ◽  
Author(s):  
Yumay Chen ◽  
Daniel J. Riley ◽  
Lei Zheng ◽  
Phang-Lang Chen ◽  
Wen-Hwa Lee
Keyword(s):  
Chromosome Segregation ◽  
Critical Role ◽  
Coiled Coil ◽  
Serine Phosphorylation ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Chromosomal Segregation ◽  
M Phase

Hec1 (highlyexpressed incancer) plays essential roles in chromosome segregation by interacting through its coiled-coil domains with several proteins that modulate the G2/M phase. Hec1 localizes to kinetochores, and its inactivation either by genetic deletion or antibody neutralization leads to severe and lethal chromosomal segregation errors, indicating that Hec1 plays a critical role in chromosome segregation. The mechanisms by which Hec1 is regulated, however, are not known. Here we show that human Hec1 is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G2/M. Nek2 phosphorylates Hec1 on serine residue 165, bothin vitroandin vivo. Yeast cells are viable without scNek2/Kin3, a close structural homolog of Nek2 that binds to both human and yeast Hec1. When the same yeasts carry an scNek2/Kin3 (D55G) or Nek2 (E38G) mutation to mimic a similar temperature-sensitivenimamutation inAspergillus, their growth is arrested at the nonpermissive temperature, because the scNek2/Kin3 (D55G) mutant binds to Hec1 but fails to phosphorylate it. Whereas wild-type human Hec1 rescues lethality resulting from deletion of Hec1 inSaccharomyces cerevesiae, a human Hec1 mutant or yeast Hec1 mutant changing Ser165to Ala or yeast Hec1 mutant changing Ser201to Ala does not. Mutations changing the same Ser residues to Glu, to mimic the negative charge created by phosphorylation, partially rescue lethality but result in a high incidence of errors in chromosomal segregation. These results suggest that cell cycle-regulated serine phosphorylation of Hec1 by Nek2 is essential for faithful chromosome segregation.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins

2016 ◽  
Vol 60 (4) ◽  
pp. 2435-2442 ◽  
Author(s):  
Tecla Ciociola ◽  
Thelma A. Pertinhez ◽  
Laura Giovati ◽  
Martina Sperindè ◽  
Walter Magliani ◽  
...  
Keyword(s):  
Self Assembly ◽  
Galleria Mellonella ◽  
Critical Role ◽  
Yeast Cells ◽  
Therapeutic Effects ◽  
Constant Region ◽  
Content Type ◽  
Complementarity Determining Regions

ABSTRACTSynthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro. The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

scholarly journals The Skn7 Response Regulator ofSaccharomyces cerevisiaeInteracts with Hsf1 In Vivo and Is Required for the Induction of Heat Shock Genes by Oxidative Stress

2000 ◽  
Vol 11 (7) ◽  
pp. 2335-2347 ◽  
Author(s):  
Desmond C. Raitt ◽  
Anthony L. Johnson ◽  
Alexander M. Erkine ◽  
Kozo Makino ◽  
Brian Morgan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Response Regulator ◽  
Coiled Coil ◽  
Normal Growth ◽  
Temperature Sensitive ◽  
Regulatory Sequences ◽  
Heat Shock Genes

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (11) ◽  
pp. 5796-5805
Author(s):  
P Orlean
Keyword(s):  
Molecular Size ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Nonpermissive Temperature ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Covalent Modifications

Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature.

scholarly journals Retinoblastoma Protein Enhances the Fidelity of Chromosome Segregation Mediated by hsHec1p

2000 ◽  
Vol 20 (10) ◽  
pp. 3529-3537 ◽  
Author(s):  
Lei Zheng ◽  
Yumay Chen ◽  
Daniel J. Riley ◽  
Phang-Lang Chen ◽  
Wen-Hwa Lee
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Cell Cycle Progression ◽  
Retinoblastoma Protein ◽  
Binding Activity ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Cycle Progression ◽  
Dna Binding Activity ◽  
M Phase

ABSTRACT Retinoblastoma protein (Rb) plays important roles in cell cycle progression and cellular differentiation. It may also participate in M phase events, although heretofore only circumstantial evidence has suggested such involvement. Here we show that Rb interacts, through an IxCxE motif and specifically during G2/M phase, with hsHec1p, a protein essential for proper chromosome segregation. The interaction between Rb and hsHec1p was reconstituted in a yeast strain in which human hsHEC1 rescues the null mutation of scHEC1. Expression of Rb reduced chromosome segregation errors fivefold in yeast cells sustained by a temperature-sensitive (ts) hshec1-113 allele and enhanced the ability of wild-type hsHec1p to suppress lethality caused by a ts smc1mutation. The interaction between Hec1p and Smc1p was important for the specific DNA-binding activity of Smc1p. Expression of Rb restored part of the inactivated function of hshec1-113p and thereby increased the DNA-binding activity of Smc1p. Rb thus increased the fidelity of chromosome segregation mediated by hsHec1p in a heterologous yeast system.

scholarly journals ROS-mediated inactivation of the PI3K/AKT pathway is involved in the antigastric cancer effects of thioredoxin reductase-1 inhibitor chaetocin

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Chuangyu Wen ◽  
Huihui Wang ◽  
Xiaobin Wu ◽  
Lu He ◽  
Qian Zhou ◽  
...  
Keyword(s):  
Critical Role ◽  
Thioredoxin Reductase ◽  
Akt Pathway ◽  
Ros Generation ◽  
In Vivo Models ◽  
M Phase ◽  
Tumorigenesis And Progression ◽  
Induced Apoptosis

Abstract Novel drugs are urgently needed for gastric cancer (GC) treatment. The thioredoxin-thioredoxin reductase (TRX-TRXR) system has been found to play a critical role in GC tumorigenesis and progression. Thus, agents that target the TRX-TRXR system may be highly efficacious as GC treatments. In this study, we showed that chaetocin, a natural product isolated from the Chaetomium species of fungi, inhibited proliferation, induced G2/M phase arrest and caspase-dependent apoptosis in both in vitro and in vivo models (cell xenografts and patient-derived xenografts) of GC. Chaetocin inactivated TRXR-1, resulting in the accumulation of reactive oxygen species (ROS) in GC cells; overexpression of TRX-1 as well as cotreatment of GC cells with the ROS scavenger N-acetyl-L-cysteine attenuated chaetocin-induced apoptosis; chaetocin-induced apoptosis was significantly increased when GC cells were cotreated with auranofin. Moreover, chaetocin was shown to inactivate the PI3K/AKT pathway by inducing ROS generation; AKT-1 overexpression also attenuated chaetocin-induced apoptosis. Taken together, these results reveal that chaetocin induces the excessive accumulation of ROS via inhibition of TRXR-1. This is followed by PI3K/AKT pathway inactivation, which ultimately inhibits proliferation and induces caspase-dependent apoptosis in GC cells. Chaetocin therefore may be a potential agent for GC treatment.

scholarly journals In Vivo Bypass of Chaperone by Extended Coiled-Coil Motif in T4 Tail Fiber

2004 ◽  
Vol 186 (24) ◽  
pp. 8363-8369 ◽  
Author(s):  
Yun Qu ◽  
Paul Hyman ◽  
Timothy Harrah ◽  
Edward Goldberg
Keyword(s):  
Bacteriophage T4 ◽  
Coiled Coil ◽  
Critical Factor ◽  
Coiled Coils ◽  
Temperature Sensitive ◽  
Tail Fiber ◽  
Type Gene ◽  
In Vivo Screen

ABSTRACT The distal-half tail fiber of bacteriophage T4 is made of three gene products: trimeric gp36 and gp37 and monomeric gp35. Chaperone P38 is normally required for folding gp37 peptides into a P37 trimer; however, a temperature-sensitive mutation in T4 (ts3813) that suppresses this requirement at 30°C but not at 42°C was found in gene 37 (R. J. Bishop and W. B. Wood, Virology 72:244-254, 1976). Sequencing of the temperature-sensitive mutant revealed a 21-bp duplication of wild-type gene 37 inserted into its C-terminal portion (S. Hashemolhosseini et al., J. Mol. Biol. 241:524-533, 1994). We noticed that the 21-amino-acid segment encompassing this duplication in the ts3813 mutant has a sequence typical of a coiled coil and hypothesized that its extension would relieve the temperature sensitivity of the ts3813 mutation. To test our hypothesis, we crossed the T4 ts3813 mutant with a plasmid encoding an engineered pentaheptad coiled coil. Each of the six mutants that we examined retained two amber mutations in gene 38 and had a different coiled-coil sequence varying from three to five heptads. While the sequences varied, all maintained the heptad-repeating coiled-coil motif and produced plaques at up to 50°C. This finding strongly suggests that the coiled-coil motif is a critical factor in the folding of gp37. The presence of a terminal coiled-coil-like sequence in the tail fiber genes of 17 additional T-even phages implies the conservation of this mechanism. The increased melting temperature should be useful for “clamps” to initiate the folding of trimeric β-helices in vitro and as an in vivo screen to identify, sequence, and characterize trimeric coiled coils.

scholarly journals Genetic and biochemical interactions between an essential kinetochore protein, Cbf2p/Ndc10p, and the CDC34 ubiquitin-conjugating enzyme.

1995 ◽  
Vol 15 (9) ◽  
pp. 4835-4842 ◽  
Author(s):  
H J Yoon ◽  
J Carbon
Keyword(s):  
Yeast Cells ◽  
Temperature Sensitive ◽  
Kinetochore Protein ◽  
Ubiquitin Conjugating Enzyme ◽  
Endogenous Substrate ◽  
Growth Phenotype ◽  
Kinetochore Function ◽  
Conjugating Enzyme

CBF2/NDC10/CTF14 encodes the 110-kDa subunit of CBF3, a key component of the yeast centromere/kinetochore. Overexpression of yeast CDC34 specifically suppresses the temperature-sensitive growth phenotype of the ndc10-1 mutation. Mutations in CDC34, which specifies a ubiquitin-conjugating enzyme, arrest yeast cells in the G1 phase of the cell cycle, with no intact spindles formed (M. G. Goebl, J. Yochem, S. Jentsch, J. P. McGrath, A. Varshavsky, and B. Byers, Science 241:1331-1335, 1988). The cdc34-2 mutation drastically alters the pattern of Cbf2p modification. Results of experiments using antibodies against Cbf2p and ubiquitin indicate that Cbf2p is ubiquitinated in vivo. Purified Cdc34p catalyzes the formation of Cbf2p-monoubiquitin conjugate in vitro. These data suggest that Cbf2p is an endogenous substrate of the CDC34 ubiquitin-conjugating enzyme and imply that ubiquitination of a kinetochore protein plays a regulatory role in kinetochore function.

scholarly journals Dual function of the NDR-kinase Dbf2 in the regulation of the F-BAR protein Hof1 during cytokinesis

2013 ◽  
Vol 24 (9) ◽  
pp. 1290-1304 ◽  
Author(s):  
Franz Meitinger ◽  
Saravanan Palani ◽  
Birgit Hub ◽  
Gislene Pereira
Keyword(s):  
Critical Role ◽  
Coiled Coil ◽  
Dual Function ◽  
Serine Residue ◽  
Bar Domain ◽  
Division Site ◽  
Coiled Coil Region ◽  
Ndr Kinase

The conserved NDR-kinase Dbf2 plays a critical role in cytokinesis in budding yeast. Among its cytokinesis-related substrates is the F-BAR protein Hof1. Hof1 colocalizes at the cell division site with the septin complex and, as mitotic exit progresses, moves to the actomyosin ring (AMR). Neither the function of Hof1 at the septin complex nor the mechanism by which Hof1 supports AMR constriction is understood. Here we establish that Dbf2 has a dual function in Hof1 regulation. First, we show that the coiled-coil region, which is adjacent to the conserved F-BAR domain, is required for the binding of Hof1 to septins. The Dbf2-dependent phosphorylation of Hof1 at a single serine residue (serine 313) in this region diminishes the recruitment of Hof1 to septins both in vitro and in vivo. Genetic and functional analysis indicates that the binding of Hof1 to septins is important for septin rearrangement and integrity during cytokinesis. Furthermore, Dbf2 phosphorylation of Hof1 at serines 533 and 563 promotes AMR constriction most likely by inhibiting the SH3-domain–dependent interactions of Hof1. Thus our data show that Dbf2 coordinates septin and AMR functions during cytokinesis through the regulation/control of Hof1.

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