Slm9, a novel nuclear protein involved in mitotic control in fission yeast

In the fission yeast Schizosaccharomyces pombe, as in other eukaryotic cells, Cdc2/cyclin B complex is the key regulator of mitosis. Perhaps the most important regulation of Cdc2 is the inhibitory phosphorylation of tyrosine-15 that is catalyzed by Wee1 and Mik1. Cdc25 and Pyp3 phosphatases dephosphorylate tyrosine-15 and activate Cdc2. To isolate novel activators of Cdc2 kinase, we screened synthetic lethal mutants in a cdc25-22 background at the permissive temperature (25 degrees ). One of the genes, slm9, encodes a novel protein of 807 amino acids. Slm9 is most similar to Hir2, the histone gene regulator in budding yeast. Slm9 protein level is constant and Slm9 is localized to the nucleus throughout the cell cycle. The slm9 disruptant is delayed at the G(2)-M transition as indicated by cell elongation and analysis of DNA content. Inactivation of Wee1 fully suppressed the cell elongation phenotype caused by the slm9 mutation. The slm9 mutant is defective in recovery from G(1) arrest after nitrogen starvation. The slm9 mutant is also UV sensitive, showing a defect in recovery from the cell cycle arrest after UV irradiation.     

The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast.

The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects. The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase. Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant. Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability. Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70.     

α-Mangostin promotes apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes by reactive oxygen species-dependent activation of ERK1/2 mitogen-activated protein kinase

α-Mangostin (α-M) is a commonly used traditional medicine with various biological and pharmacological activities. Our study aimed to explore the effects and mechanism of α-M in regulating apoptosis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). α-M of 10 to 100 μM was used to treat RA-FLS for 24 hours, followed by measuring cell viability and apoptosis. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases was detected. Treatment of α-M promoted apoptosis and reduced viability of RA-FLS in a dose-dependent manner. The mitochondrial membrane potential in RA-FLS was remarkably reduced by α-M treatment, accompanied by the cytochrome c accumulation in the cytosol and increased activities of caspase-3 and caspase-9. Moreover, we found that α-M treatment promoted ROS production and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. The proapoptotic activity of α-M in RA-FLS was markedly reversed by the co-induction with the ERK1/2 inhibitor LY3214996 or ROS scavenger N-acetyl-l -cysteine. In conclusion, our studies found that α-M had remarkable proapoptotic activities in RA-FLS, which is regulated by the induction of ROS accumulation and ERK1/2 phosphorylation. α-M may thus have potential therapeutic effects for rheumatoid arthritis.     

Male germ line development in Arabidopsis. duo pollen mutants reveal gametophytic regulators of generative cell cycle progression

Male germ line development in flowering plants is initiated with the formation of the generative cell that is the progenitor of the two sperm cells. While structural features of the generative cell are well documented, genetic programs required for generative cell cycle progression are unknown. We describe two novel Arabidopsis (Arabidopsis thaliana) mutants, duo pollen1 (duo1) and duo pollen2 (duo2), in which generative cell division is blocked, resulting in the formation of bicellular pollen grains at anthesis. duo1 and duo2 map to different chromosomes and act gametophytically in a male-specific manner. Both duo mutants progress normally through the first haploid division at pollen mitosis I (PMI) but fail at distinct stages of the generative cell cycle. Mutant generative cells in duo1 pollen fail to enter mitosis at G2-M transition, whereas mutant generative cells in duo2 enter PMII but arrest at prometaphase. In wild-type plants, generative and sperm nuclei enter S phase soon after inception, implying that male gametic cells follow a simple S to M cycle. Mutant generative nuclei in duo1 complete DNA synthesis but bypass PMII and enter an endocycle during pollen maturation. However, mutant generative nuclei in duo2 arrest in prometaphase of PMII with a 2C DNA content. Our results identify two essential gametophytic loci required for progression through different phases of the generative cell cycle, providing the first evidence to our knowledge for genetic regulators of male germ line development in flowering plants.     

mad—overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Mad protein has been shown as an antagonist of cMyc protein in some cell lines.The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally.An eukarryotic vector pCDNA Ⅲ containing full ORF fragment of mad cDNA was transfected into targeted cells.Under G418 selection,stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells.DNA synthesis,cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cells were partially inhibited in comparison to control cells.Flos cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase,resulting in the retardation of cell proliferation.RT-PCR detected a marked inhibition of the expression of cdc25A,an important regulator gene of G0/G1 to S phase in cell cycle.It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-74040M1 cells in the absence of serume.Thus,Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.     

The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression

Cell cycle growth arrest is an important cellular response to genotoxic stress. Gadd45, a p53-regulated stress protein, plays an important role in the cell cycle G(2)-M checkpoint following exposure to certain types of DNA-damaging agents such as UV radiation and methylmethane sulfonate. Recent findings indicate that Gadd45 interacts with Cdc2 protein and inhibits Cdc2 kinase activity. In the present study, a series of Myc-tagged Gadd45 deletion mutants and a Gadd45 overlapping peptide library were used to define the Gadd45 domains that are involved in the interaction of Gadd45 with Cdc2. Both in vitro and in vivo studies indicate that the interaction of Gadd45 with Cdc2 involves a central region of the Gadd45 protein (amino acids 65-84). The Cdc2-binding domain of Gadd45 is also required for Gadd45 inhibition of Cdc2 kinase activity. Sequence analysis of the central Gadd45 region reveals no homology to inhibitory motifs of known cyclin-dependent kinase inhibitors, indicating that the Cdc2-binding and -inhibitory domains on Gadd45 are a novel motif. The peptide containing the Cdc2-binding domain (amino acids 65-84) disrupted the Cdc2-cyclin B1 protein complex, suggesting that dissociation of this complex results from a direct interaction between the Gadd45 and Cdc2 proteins. GADD45-induced cell cycle G(2)-M arrest was abolished when its Cdc2 binding motif was disrupted. Importantly, a short term survival assay demonstrated that GADD45-induced cell cycle G(2)-M arrest correlates with GADD45-mediated growth suppression. These findings indicate that the cell cycle G(2)-M growth arrest mediated by GADD45 is one of the major mechanisms by which GADD45 suppresses cell growth.     

The novel DNA damage checkpoint protein ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast.

The DDC1 gene was identified, together with MEC3 and other checkpoint genes, during a screening for mutations causing synthetic lethality when combined with a conditional allele altering DNA primase. Deletion of DDC1 causes sensitivity to UV radiation, methyl methanesulfonate (MMS) and hydroxyurea (HU). ddc1Delta mutants are defective in delaying G1-S and G2-M transition and in slowing down the rate of DNA synthesis when DNA is damaged during G1, G2 or S phase, respectively. Therefore, DDC1 is involved in all the known DNA damage checkpoints. Conversely, Ddc1p is not required for delaying entry into mitosis when DNA synthesis is inhibited. ddc1 and mec3 mutants belong to the same epistasis group, and DDC1 overexpression can partially suppress MMS and HU sensitivity of mec3Delta strains, as well as their checkpoint defects. Moreover, Ddc1p is phosphorylated periodically during a normal cell cycle and becomes hyperphosphorylated in response to DNA damage. Both phosphorylation events are at least partially dependent on a functional MEC3 gene.     

Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae.

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.     

敲除 Sam68 基因导致白血病细胞系细胞生长迟缓和 G2/M 期延长

RNA 结合蛋白 Sam68 是细胞有丝分裂期 Src 酪氨酸磷酸化的靶蛋白 . 尽管确切机制尚不清楚,一些人还是认为 Sam68 可通过调控 RNA 的代谢参与细胞周期调控 . 利用基因打靶技术,在 DT40 细胞分离出 Sam68 基因缺失的细胞系 . 利用该细胞系,进行 Sam68 的功能解析 . 与野生型细胞系相比, Sam68 基因缺失细胞表现出明显的生长速度迟缓 . 通过细胞周期研究揭示 , 这些细胞生长速度延迟是由于细胞周期中的 G2/M 期延长 . 因为参与细胞周期 G2/M 期调控的周期因子 Cdc2 激酶的活性没有改变,所以提示 Sam68 不依赖于 Cdc2 激酶的活性参与细胞周期中 G2/M 期调控 .     

A genome–wide screen to identify genes controlling the rate of entry into mitosis in fission yeast

We have carried out a haploinsufficiency (HI) screen in fission yeast using heterozygous deletion diploid mutants of a genome-wide set of cell cycle genes to identify genes encoding products whose level determines the rate of progression through the cell cycle. Cell size at division was used as a measure of advancement or delay of the G2-M transition of rod-shaped fission yeast cells. We found that 13 mutants were significantly longer or shorter (greater than 10%) than control cells at cell division. These included mutants of the cdc2, cdc25, wee1 and pom1 genes, which have previously been shown to play a role in the timing of entry into mitosis, and which validate this approach. Seven of these genes are involved in regulation of the G2-M transition, 5 for nuclear transport and one for nucleotide metabolism. In addition we identified 4 more genes that were 8–10% longer or shorter than the control that also had roles in regulation of the G2-M transition or in nuclear transport. The genes identified here are all conserved in human cells, suggesting that this dataset will be useful as a basis for further studies to identify rate-limiting steps for progression through the cell cycle in other eukaryotes.     

In vivo characterization of the nonessential budding yeast anaphase-promoting complex/cyclosome components Swm1p, Mnd2p and Apc9p

We have examined the in vivo requirement of two recently identified nonessential components of the budding yeast anaphase-promoting complex, Swm1p and Mnd2p, as well as that of the previously identified subunit Apc9p. swm1Delta mutants exhibit synthetic lethality or conditional synthetic lethality with other APC/C subunits and regulators, whereas mnd2Delta mutants are less sensitive to perturbation of the APC/C. swm1Delta mutants, but not mnd2Delta mutants, exhibit defects in APC/C substrate turnover, both during the mitotic cell cycle and in alpha-factor-arrested cells. In contrast, apc9Delta mutants exhibit only minor defects in substrate degradation in alpha-factor-arrested cells. In cycling cells, degradation of Clb2p, but not Pds1p or Clb5p, is delayed in apc9Delta. Our findings suggest that Swm1p is required for full catalytic activity of the APC/C, whereas the requirement of Mnd2p for APC/C function appears to be negligible under standard laboratory conditions. Furthermore, the role of Apc9p in APC/C-dependent ubiquitination may be limited to the proteolysis of a select number of substrates.     

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2A^Cdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.     

Modulation of expression of the stress-inducible p118 of Saccharomyces cerevisiae by cAMP. II. A study of p118 expression in mutants of the cAMP cascade

In the preceding paper, we have identified a protein of Mr = 118,000 which is induced by stress conditions that lead to cessation of DNA synthesis and cell division (Verma, R., Iida, H., and Pardee, A.B. (1988) J. Biol. Chem. 263, 8569-8575). In the current study, we have investigated the possible role this protein may play in cellular proliferation by studying p118 expression in mutants of the cAMP metabolic pathway. The cyr 1-2 mutant gene encodes a thermolabile adenylate cyclase whose activity is only 7% of wild type even at permissive temperatures (23 degrees C). We have found that at 23 degrees C, the G1 period was 5-fold longer in cyr 1-2 than in CYR1+ cells and that p118 was constitutively expressed in these slow cycling mutants. Addition of 8-bromo-cAMP to cyr 1-2 mutants restored growth at both the restrictive and permissive temperatures and resulted in a shut-off in the synthesis of p118. The effect of the analog on p118 expression was rapid, preceding the increase in cell number and percentage-budded cells. In contrast to wild type cells, p118 synthesis was not induced by sulfur starvation in RAS2val19 mutants possessing high levels of adenylate cyclase activity and bcy1 mutants defective in the regulatory subunit of cAMP-dependent protein kinase. A large body of evidence exists supporting a role of cAMP in positive control of cell proliferation. It is therefore possible that conditions which decrease cAMP arrest growth through a chain of events that include p118 induction.     

Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites

Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P(2) production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.