Dbf2–Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis

Exit from mitosis is characterized by a precipitous decline in cyclin-dependent kinase (Cdk) activity, dissolution of mitotic structures, and cytokinesis. In Saccharomyces cerevisiae, mitotic exit is driven by a protein phosphatase, Cdc14, which is in part responsible for counteracting Cdk activity. Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown. In this study, we show that a MEN component, protein kinase Dbf2–Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity. Our results define a mechanism by which the MEN promotes exit from mitosis

Predisposition to Cancer Caused by Genetic and Functional Defects of Mammalian Atad5

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5+/m) mice that were haploinsuffficient for Atad5. Atad5+/m mice displayed high levels of genomic instability in vivo, and Atad5+/m mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5+/m mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5+/m mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis

Regulation of eukaryotic dna replication at the initiation step

The studies of cell growth and division have remained at the centre of biomedical research for more than 100 years. The combination of genetic, biochemical, molecular and cell biological techniques recently yielded a burst in what is known of the molecular control of cell growth processes. The initiation of DNA replication is crucial for the stability of the genetic information of a cell. Two factors, Cdc45p (cell division cycle 45p) and DNA polymerase a-primase, are necessary in this process. Depending on growth signals, Cdc45p is expressed as a late protein. New phosphorylation-specific antibodies specifically recognize the phosphorylated subunit, p68, of the four subunit DNA polymerase a-primase and show that the phosphorylated polypeptide is exclusively nuclear

The Salmonella Type III Effector SspH2 Specifically Exploits the NLR Co-chaperone Activity of SGT1 to Subvert Immunity

To further its pathogenesis, S. Typhimurium delivers effector proteins into host cells, including the novel E3 ubiquitin ligase (NEL) effector SspH2. Using model systems in a cross-kingdom approach we gained further insight into the molecular function of this effector. Here, we show that SspH2 modulates innate immunity in both mammalian and plant cells. In mammalian cell culture, SspH2 significantly enhanced Nod1-mediated IL-8 secretion when transiently expressed or bacterially delivered. In addition, SspH2 also enhanced an Rx-dependent hypersensitive response in planta. In both of these nucleotide-binding leucine rich repeat receptor (NLR) model systems, SspH2-mediated phenotypes required its catalytic E3 ubiquitin ligase activity and interaction with the conserved host protein SGT1. SGT1 has an essential cell cycle function and an additional function as an NLR co-chaperone in animal and plant cells. Interaction between SspH2 and SGT1 was restricted to SGT1 proteins that have NLR co-chaperone function and accordingly, SspH2 did not affect SGT1 cell cycle functions. Mechanistic studies revealed that SspH2 interacted with, and ubiquitinated Nod1 and could induce Nod1 activity in an agonist-independent manner if catalytically active. Interestingly, SspH2 in vitro ubiquitination activity and protein stability were enhanced by SGT1. Overall, this work adds to our understanding of the sophisticated mechanisms used by bacterial effectors to co-opt host pathways by demonstrating that SspH2 can subvert immune responses by selectively exploiting the functions of a conserved host co-chaperone

SspH2 stability and activity are enhanced by HsSGT1A <i>in vitro</i>.

<p><b>A,</b> Time course of <i>in vitro</i> ubiquitination assay conducted with purified recombinant HA-Ubiquitin, UBE1 (E1), UbcH5b (E2), His-HsSGT1A and GST-HA-SspH2 as indicated. Reactions were quenched at the indicated time and immunoblotted with the indicated antibodies to detect SspH2 activity, SspH2 stability and HsSGT1A ubiquitination. <b>B, </b><i>In vitro</i> ubiquitination assay reaction components were pre-incubated for the indicated times, initiated with HA-ubiquitin and quenched after 2 hours. Samples were immunoblotted as outlined above. Molecular weights are indicated on the left.</p

<i>S.</i> Typhimurium SspH2 does not alter the essential cell cycle function of SGT1.

<p><b>A,</b> Dilution series of wild type yeast expressing empty vector (EV) or SspH2 with HA-epitope tag grown at 25°C. The expression of HA-tagged SspH2 was assessed by immunoblot in total lysate. <b>B,</b> Dilution series of yeast <i>sgt1-3</i> complemented with ScSgt1p (Sc), HsSGT1A (HsA), HsSGT1B (HsB) or empty vector (EV) and expressing SspH2-HA (+) or empty vector (−), were grown at the permissive (25°C) and restrictive (32°C) temperatures. <b>C,</b> The expression of HA-tagged SspH2 in cultures from (<b>B</b>) was assessed by immunoblot in total cell lysate. The membrane was stained with Ponceau S (Pon. S) to indicate protein loading. <b>D,</b> HeLa cells transfected with plasmid control (EV) or constitutive expression vectors for SspH1 or SspH2 were quantified for cytotoxicity. Data are presented as the mean ± standard error of the mean for three independent determinations. <i>S.</i> Typhimurium effectors were tagged with an HA epitope and shown in the inset is the expression of transfected constructs as assessed by α-HA IB. Calnexin is a loading control.</p

<i>S.</i> Typhimurium SspH2 enhances SGT1-dependent NLR immune response in mammalian cells.

<p><b>A</b>, Establishment of a Nod1-mediated IL-8 secretion assay in HeLa cells. Secreted IL-8 was quantified in the presence and absence of transfected Nod1 and Nod1 agonist. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003518#s4" target="_blank">Materials and methods</a> for more detail. <b>B</b>, Effect of <i>S.</i> Typhimurium effector constructs on Nod1-mediated IL-8 secretion. HeLa cells were co-transfected with Nod1 and effector constructs and treated with Nod1 agonist. Data are presented as the mean ± standard error of the mean for three (<b>A</b>) and twelve (<b>B</b>) independent determinations. Data were analyzed using a non-parametric Mann-Whitney test and * denotes <i>p</i><0.005 between the indicated sample and the empty vector control (EV) sample. # denotes <i>p</i><0.005 between the indicated samples. <b>C</b>, The expression of epitope tagged <i>S.</i> Typhimurium effectors (HA) and exogenous Nod1 (FLAG) was assessed by immunoblot in total lysate. Calnexin is a loading control. <b>D</b>, Effect of <i>S.</i> Typhimurium infection on IL-8 secretion. HeLa cells were infected with the wild type strain, <i>sspH2</i> deletion mutant (Δ<i>sspH2</i>), and <i>sspH2</i> deletion mutant complemented with empty vector (Δ<i>sspH2</i>+pWSK) or <i>sspH2</i> (Δ<i>sspH2</i>+p<i>sspH</i>2-HA). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003518#s4" target="_blank">Materials and methods</a> for more detail. Data are presented as the mean ± standard error of the mean for six independent determinations. Data were analyzed using a non-parametric Mann-Whitney test and * denotes <i>p</i><0.005 between the indicated sample and all other samples. ns, denotes no significant difference between the indicated samples.</p

<i>S.</i> Typhimurium SspH2 enhances SGT1-dependent NLR immune response <i>in planta</i>.

<p><b>A</b>, An NbSGT1-silenced (TRV:SGT) Rx-transgenic <i>N. benthamiana</i> (Nb Rx) leaf was agro-infiltrated with PVX-GFP and also 5xMyc-AtSGT1A (AtA), GUS, SspH2-3xFLAG (H2), SspH1-3xFLAG (H1) and SspH2C580A-3xFLAG (C580A) as indicated. As controls the same infiltrations were performed in a control-silenced (TRV:00) Nb Rx leaf and non-transgenic (Nb WT) TRV:SGT leaf. Leaves were imaged under UV lighting 7 days post-infiltration. <b>B</b>, Infiltration site lysate from samples in (<b>A</b>) were immunoblotted with α-GFP to detect PVX replication. Membranes were stained with Ponceau S (Pon. S) to indicate protein loading. <b>C</b>, Nb Rx TRV:SGT leaf was agro-infiltrated as indicated in (<b>A</b>) and imaged under brightfield 7 days post-infiltration. Higher magnification panels of the brightfield image are provided for comparison.</p