A Drosophila gene encoding a DEAD box RNA helicase can suppress loss of wee1/mik1 function in Schizosaccharomyces pombe

We describe a screen to isolate cDNAs encoding Drosophila mitosis inhibitors capable of suppressing the mitotic catastrophe phenotype resulting in Schizosaccharomyces pombe from the combination of the weel-50 mutation with either a deletion allele of mil1, or with overexpression of cdc25 ⁺. One plasmid was isolated which could suppress the temperature sensitive lethality of both these strains. The cDNA in this plasmid encodes a protein highly homologous to the DEAD-box family of ATP-dependent RNA helicases, rather than to protein kinases as might be expected. It is possible that the RNA helicase described here may regulate entry into mitosis by down regulating the expression of other genes whose activity may be rate-limiting for entry into mitosis.     

Fission Yeast dim1+ Encodes a Functionally Conserved Polypeptide Essential for Mitosis

In a screen for second site mutations capable of reducing the restrictive temperature of the fission yeast mutant cdc2-D217N, we have isolated a novel temperature-sensitive mutant, dim1-35. When shifted to restrictive temperature, dim1-35 mutant cells arrest before entry into mitosis or proceed through mitosis in the absence of nuclear division, demonstrating an uncoupling of proper DNA segregation from other cell cycle events. Deletion of dim1 from the Schizosaccharomyces pombe genome produces a lethal G₂ arrest phenotype. Lethality is rescued by overexpression of the mouse dim1 homolog, mdim1. Likewise, deletion of the Saccharomyces cerevisiae dim1 homolog, CDH1, is lethal. Both mdim1 and dim1⁺ are capable of rescuing lethality in the cdh1::HIS3 mutant. Although dim1-35 displays no striking genetic interactions with various other G₂/M or mitotic mutants, dim1-35 cells incubated at restrictive temperature arrest with low histone H1 kinase activity. Morevoer, dim1-35 displays sensitivity to the microtubule destabilizing drug, thiabendazole (TBZ). We conclude that Dim1p plays a fundamental, evolutionarily conserved role as a protein essential for entry into mitosis as well as for chromosome segregation during mitosis. Based on TBZ sensitivity and failed chromosome segregation in dim1-35, we further speculate that Dim1p may play a role in mitotic spindle formation and/or function.     

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser²⁰⁵⁶, Thr²⁶⁴⁷ and Thr²⁶⁰⁹ in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser³²⁰⁵ and Thr³⁹⁵⁰ in mitosis. Phosphorylation of Thr³⁹⁵⁰ is DNA-PK-dependent, whereas phosphorylation of Ser³²⁰⁵ requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser³²⁰⁵ in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser³²⁰⁵ in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr⁶⁸ in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs’ role in mitosis may be mechanistically distinct from its well-established role in NHEJ.     

Effect of serine/threonine protein kinases and protein phosphatases inhibitors on mitosis progression in a synchronized tobacco BY-2 culture

In order to investigate the role of various serine/threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells, the influence of cyclin(olomoucine) and Ca²⁺/calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine), and a protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin-dependent protein kinases and protein kinase C caused a prophase delay, reduced the mitotic index, and displaced the mitotic peak as compared with control cells. Inhibition of Ca²⁺/calmodulin-dependent protein kinases enhanced the cells entry into prophase and delayed their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances synchronized BY-2 cells entering into all phases of mitosis.     

Budding Yeast Greatwall and Endosulfines Control Activity and Spatial Regulation of PP2ACdc55 for Timely Mitotic Progression

Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2A^Cdc55 phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2) bind to PP2A^Cdc55 in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2A^Cdc55 activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like PP2A^Cdc55, timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2A^Cdc55, which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms.     

Budding Yeast Swe1 Is Involved in the Control of Mitotic Spindle Elongation and Is Regulated by Cdc14 Phosphatase during Mitosis

Cyclin-dependent kinase (Cdk1) activity is required for mitotic entry, and this event is restrained by an inhibitory phosphorylation of the catalytic subunit Cdc28 on a conserved tyrosine (Tyr¹⁹). This modification is brought about by the protein kinase Swe1 that inhibits Cdk1 activation thus blocking mitotic entry. Swe1 levels are regulated during the cell cycle, and they decrease during G₂/M concomitantly to Cdk1 activation, which drives entry into mitosis. However, after mitotic entry, a pool of Swe1 persists, and we collected evidence that it is involved in controlling mitotic spindle elongation. We also describe that the protein phosphatase Cdc14 is implicated in Swe1 regulation; in fact, we observed that Swe1 dephosphorylation in vivo depends on Cdc14 that, in turn, is able to control its subcellular localization. In addition we show that the lack of Swe1 causes premature mitotic spindle elongation and that high levels of Swe1 block mitotic spindle elongation, indicating that Swe1 inhibits this process. Importantly, these effects are not dependent upon the role of in Cdk1 inhibition. These data fit into a model in which Cdc14 binds and inhibits Swe1 to allow timely mitotic spindle elongation.     

Group II intron-based gene targeting reactions in eukaryotes

Background

Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors (“targetrons”) with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes.

Methodology/Principal Findings

By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg²⁺ concentrations. By supplying additional Mg²⁺, site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg²⁺-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization.

Conclusions/Significance

Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms.     

RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens. The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5′-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3I nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5′-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5′-terminal region of RNA1 is crucial for its function.     

Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli

The ssb-1 gene encoding a mutant Escherichia coli single-stranded DNA-binding protein has been cloned into plasmid pACYC184. The amount of overproduction of the cloned ssb-1 gene is dependent upon its orientation in the plasmid. In the less efficient orientation, 25-fold more mutant protein is produced than in strains carrying only one (chromosomal) copy of the gene: the other orientation results in more than 60-fold overproduction of this protein. Analysis of the effects of overproduction of the ssb-1 encoded protein has shown that most of the deficiencies associated with the ssb-1 mutation when present in single gene copy, including temperature-sensitive conditional lethality and deficiencies in amplified synthesis of RecA protein and ultraviolet light-promoted induction of prophage λ⁺, are reversed by increased production of ssb-1 mutant protein. These results provide evidence in vivo that SSB protein plays an active role in recA-dependent processes. Homogenotization of a nearby genetic locus (uvrA) was identified in the cloning of the ssb-1 mutant gene. This observation has implications in the analysis of uvrA^? mutant strains and will provide a means of transferring ssb^? mutations from plasmids to the chromosome. On a broader scale, the observation may provide the basis of a general strategy to transfer mutations between plasmids and chromosomes.     

Reversible tyrosine phosphorylation and cell cycle control

In eukaryotic organisms, reversible tyrosine phosphorylation has been established as an important element in the regulation of cell growth and more recently as an essential element in the regulation of the cell division cycle. The activity of p34^cdc2, a protein kinase whose activity is required for the entry of cells into mitosis, is tightly controlled by reversible phosphorylation at tyrosine 15. A complex network of interacting protein kinases and protein phosphatases regulate the state of p34^cdc2 tyrosine phosphorylation and therefore the entry of cells into mitosis. In the fission yeast Schizosaccharomyces pombe, genes encoding several of these protein kinases and protein phosphatases have been obtained through genetic approaches. In this review, we will focus on the protein kinases encoded by wee1⁺, mik1⁺ and cdr1⁺/nim1⁺ and the protein phosphatases encoded by cdc25⁺ and pyp1⁺, pyp2⁺ and pyp3⁺. Homologs of many of these regulators have been identified and characterized in higher eukaryotes underscoring the importance of reversible tyrosine phosphorylation as a universal mechanism for the regulation of the cell division cycle.     

Dynein intermediate chain 2c (DNCI2c) complex is essential for exiting Mad2-dependent spindle assembly checkpoint

The Mad2 protein plays a key role in the spindle assembly checkpoint (SAC) function. The SAC pathway delays mitotic progression into anaphase until all kinetochores attach to the spindle during mitosis. The formation of the Mad2–p31^comet complex correlates with the completion of spindle attachment and the entry into anaphase during mitosis.Herein, we showed that dynein intermediate chain 2c (DNCI2c)—a subunit of dynein motor protein—forms an immunocomplex with p31^comet during mitosis. DNCI2c-knockdown resulted in prolonged mitotic arrest in a Mad2-dependent manner. Furthermore, DNCI2c-knockdown-induced mitotic arrest was not rescued by p31^comet overexpression. However, the combination of p31^comet overexpression with the mitotic drug treatment reversed the mitotic arrest in DNCI2c-knockdown. Together, these results indicate that the DNCI2c–p31^comet complex plays an important role in exiting Mad2-dependent SAC.     

The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis

The protein kinase p34^cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34^CDC2 protein kinase from chicken. The response of the chicken p34^CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34^CDC2 protein divide at reduced size at 31°?C. Cells are temperature sensitive at 35.5°?C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34^CDC2 are cold sensitive. At 19°?C to 25°?C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34^CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest.     

A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na⁺ and H⁺ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca²⁺. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.     

A phosphorylation site mutant of Schizosaccharomyces pombe cdc2p fails to promote the metaphase to anaphase transition

The protein kinase cdc2p is a key regulator of the G1-S and G2-M cell cycle transitions in the yeast Schizosaccharomyces pombe. Activation of cdc2p is regulated by its phosphorylation state and by interaction with other proteins. We have analyzed the consequences for cell cycle progression of altering the conserved threonine phosphorylation site, within the activation loop of cdc2p, to glutamic acid. This mutant, T167 E, promotes entry into mitosis, as judged by the accumulation of mitotic spindles and condensed chromosomes, despite the fact that it lacks demonstrable kinase activity both in vitro and in vivo. However, T167 E cannot promote the metaphase-anaphase transition. Since a component of the anaphase-promoting complex (APC) in S. pombe, cut9p, remains hypophosphorylated at the T167 E arrest point, the cell cycle block might be due to the inability of T167 E to activate the APC. T167 E is lethal when overexpressed, and overproduction also causes a mitotic arrest. Multicopy suppressors of the dominant negative phenotype were isolated, and identified as cdc13 ⁺ and suc1 ⁺ . Overexpression of suc1 ⁺ suppresses the effects of T167 E overproduction by restoring sufficient amounts of suc1p to the cell to allow passage through mitosis.     

Identification of a Novel Function of PiT1 Critical for Cell Proliferation and Independent of Its Phosphate Transport Activity

PiT1 is a Na⁺-phosphate (P_i) cotransporter located at the plasma membrane that enables P_i entry into the cell. Its broad tissue expression pattern has led to the idea that together with the closely related family member PiT2, PiT1 is the ubiquitous supplier of P_i to the cell. Moreover, the role of P_i in phosphorylation reactions, ATP production, DNA structure, and synthesis has led to the view that P_i availability could be an important determinant of cell growth. However, these issues have not been clearly addressed to date, and the role of either P_i or PiT proteins in cell proliferation is unknown. Using RNA interference in HeLa and HepG2 cells, we show that transient or stable PiT1 depletion markedly reduces cell proliferation, delays cell cycle, and impairs mitosis and cytokinesis. In vivo, PiT1 depletion greatly reduced tumor growth when engineered HeLa cells were injected into nude mice. We provide evidence that this effect on cell proliferation is specific to PiT1 and not shared by PiT2 and is not the consequence of impaired membrane Na⁺-P_i transport. Moreover, we show that modulation of cell proliferation by PiT1 is independent from its transport function because the proliferation of PiT1-depleted cells can be rescued by non-transporting PiT1 mutants. PiT1 depletion leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase, whereas other MAP kinases and downstream targets of mammalian target of rapamycin (mTOR) remain unaffected. This study is the first to describe the effects of a P_i transporter in cell proliferation, tumor growth, and cell signaling.