Anaphase-promoting Complex Subunit Research Articles

Inactivation and Disassembly of the Anaphase-Promoting Complex during Human Cytomegalovirus Infection Is Associated with Degradation of the APC5 and APC4 Subunits and Does Not Require UL97-Mediated Phosphorylation of Cdh1

Infection of quiescent cells by human cytomegalovirus (HCMV) elicits severe cell cycle deregulation, resulting in a G(1)/S arrest, which can be partly attributed to the inactivation of the anaphase-promoting complex (APC). As we previously reported, the premature phosphorylation of its coactivator Cdh1 and/or the dissociation of the core complex can account for the inactivation. We have expanded on these results and further delineated the key components required for disabling the APC during HCMV infection. The viral protein kinase UL97 was hypothesized to phosphorylate Cdh1, and consistent with this, phosphatase assays utilizing a virus with a UL97 deletion mutation (ΔUL97 virus) indicated that Cdh1 is hypophosphorylated at early times in the infection. Mass spectrometry analysis demonstrated that UL97 can phosphorylate Cdh1 in vitro, and the majority of the sites identified correlated with previously characterized cyclin-dependent kinase (Cdk) consensus sites. Analysis of the APC core complex during ΔUL97 virus infection showed APC dissociation occurring at the same time as during infection with wild-type virus, suggesting that the UL97-mediated phosphorylation of Cdh1 is not required for this to occur. Further investigation of the APC subunits showed a proteasome-dependent loss of the APC5 and APC4 subunits that was temporally associated with the disassembly of the APC. Immediate early viral gene expression was not sufficient for the degradation of APC4 and APC5, indicating that a viral early gene product(s), possibly in association with a de novo-synthesized cellular protein(s), is involved.

Abstract 2062: A quantitative proteomic investigation of microRNA-145 in breast cancer

Abstract MicroRNAs (miRNAs) represent an endogenous, post-transcriptional gene expression regulatory mechanism that is mediated by small, non-coding double-stranded RNAs (dsRNA) which target discrete messenger RNAs (mRNAs) resulting in their degradation or translational repression. Current computational methodologies predict that an average of 200 unique genes may be targeted by a single miRNA with the vast majority remaining, as yet, experimentally unvalidated. Furthermore, as miRNAs regulate their gene targets post-transcriptionally, the application of global, quantitative proteomic analysis to miRNA target discovery will yield a more comprehensive assessment of miRNA activity than gene expression analysis alone. Several miRNAs have been shown to be robustly downregulated in breast cancer, including microRNA-145 (miR-145). Recent investigations of miR-145 in breast cancer have revealed tumor suppressor activities for this miRNA and that loss of miR-145 expression may be associated with early stages of breast cancer pathogenesis. To elucidate targets of miR-145 and its role in breast cancer, we restored expression in the human breast cancer cell lines MDA-MB-231 and SKBR3 and conducted both phenotypic and global, quantitative protein expression analysis utilizing mass spectrometry-based proteomics. Phenotypic analyses indicated that MDA-MB-231 and SKBR3 cells stably expressing miR-145 exhibit a growth advantage relative to control cells. In support of this evidence, GO enrichment analysis of significantly differentially expressed proteins in miR-145-expressing MDA-MB-231 (MDA-145) cells revealed modulation of factors associated with cell cycle regulation, such as increased expression of the G2/M-phase regulatory factors anaphase promoting complex subunit 1 (ANAPC1) and cell division cycle 20 homolog (CDC20) expression and decreased expression of DnaJ (Hsp40) homolog (DNAJA3). Investigation of miR-145-specific effects via sequence analysis of 3’ untranslated regions derived from genes corresponding to proteins which were decreased in abundance in MDA-145 cells revealed 33 target candidates that contained a miR-145 “seed” motif. Of these 33 candidates, 21 have been previously predicted as miR-145 targets (miRNAMap). Target candidates are associated with cell cycle regulation and mitochondrial function and include integrin linked kinase (ILK), the cytokine growth factor chromosome 19 open reading frame 10 (C19ORF10) and microsomal glutathione S-transferase 1 (MGST1). Validation of target candidates and exploration of their role in the miR-145-mediated growth advantage phenotype are currently underway. In conclusion, these results indicate that restoration of stable miR-145 expression in breast cancer cells confers a growth advantage which is underscored by modulation of proteins associated with cell cycle regulation and mitochondrial activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2062.

Isolation and characterization of genes functionally involved in ovarian development of the giant tiger shrimp Penaeus monodon by suppression subtractive hybridization (SSH)

Suppression subtractive hybridization (SSH) libraries between cDNA in stages I (previtellogenic) and III (cortical rod) ovaries of the giant tiger shrimp (Penaeus monodon) were established. In all, 452 ESTs were unidirectionally sequenced. Sequence assembly generated 28 contigs and 201 singletons, 109 of which (48.0%) corresponding to known sequences previously deposited in GenBank. Several reproduction-related transcripts were identified. The full-length cDNA of anaphase promoting complex subunit 11 (PmAPC11; 600 bp with an ORF of 255 bp corresponding to a polypeptide of 84 amino acids) and selenoprotein Mprecursor (PmSePM; 904 bp with an ORF of 396 bp corresponding to a polypeptide of 131 amino acids) were characterized and reported for the first time in penaeid shrimp. Semiquantitative RT-PCR revealed that the expression levels of PmSePM and keratinocyte-associated protein 2 significantly diminished throughout ovarian development, whereas Ser/Thrcheckpoint kinase 1 (Chk1), DNA replication licensing factor mcm2 and egalitarian were down-regulated in mature ovaries of wild P. monodon (p < 0.05). Accordingly, the expression profiles of PmSePM and keratinocyte-associated protein 2 could be used as biomarkers for evaluating the degree of reproductive maturation in domesticated P. monodon.

Cell cycle deregulation by a poxvirus partial mimic of anaphase-promoting complex subunit 11.

The anaphase-promoting complex (APC), or cyclosome, is a ubiquitin ligase with major roles in cell cycle regulation. It is required for mitotic exit, but must be deactivated for the G(1)/S phase transition to occur. APC consists of at least 12 subunits with the catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. APC11 facilitates ubiquitin chain formation by recruiting ubiquitin-charged conjugating enzymes through its RING-H2 domain. We report that a small number of poxviruses encode RING-H2 proteins with sequence similarities to APC11. We show that a representative of these viral proteins mimics APC11 in its interactions with APC, but unlike APC11, the viral protein fails to promote ubiquitin chain formation. This absence of ubiquitin ligase activity is linked to a distinctive sequence variation within its RING-H2 domain. Expression of the viral protein led to cell cycle deregulation and the accumulation of APC substrates in a manner consistent with impaired APC function. Our data characterize this protein as a regulator of APC activity, and consequently, we have called it PACR (poxvirus APC/cyclosome regulator). Deletion of the PACR gene substantially reduced viral replication. Here, we report a viral mimic of an APC component and reveal an intriguing mechanism by which viruses can manipulate cell cycle progression and, thereby, promote their own replication.

Overexpression of the Arabidopsis anaphase promoting complex subunit CDC27a increases growth rate and organ size

The Anaphase Promoting Complex (APC) controls CDK activity by targeting the ubiquitin-dependent proteolysis of S-phase and mitosis-promoting cyclins. Here, we report that the ectopic expression of the Arabidopsis CDC27a, an APC subunit, accelerates plant growth and results in plants with increased biomass production. CDC27a overexpression was associated to apical meristem restructuration, protoplasts with higher (3)H-thimidine incorporation and altered cell-cycle marker expression. Total protein extracts immunoprecipitated with a CDC27a antibody showed ubiquitin ligase activity, indicating that the Arabidopsis CDC27a gets incorporated into APC complexes. These results indicate a role of AtCDC27a in regulation of plant growth and raise the possibility that the activity of the APC and the rates of plant cell division could be regulated by the concentration of the CDC27a subunit.

The Mechanism by Which Fizzy-Related (Fzr1) Controls Prophase I Arrest in Mouse Oocytes.

Recently we discovered that Fzr1/Cdh1 activity is essential to maintain prophase I stage arrest in fully-grown mouse oocytes by mediating degradation of cyclin B1. Fzr1 is an activator on the Anaphase-Promoting Complex (APC), a multisubunit E3 ubiquitin ligase responsible for polyubiquitinating substrates, such as cyclin B1, that in so being are earmarked for degradation through the 26S proteasome. Cyclin B1 is the regulatory subunit of Maturation-Promoting Factor, forming a heterodimer with CDK1, whose kinase activity requires cyclin B1 binding and is essential for dissolution of the nuclear envelope and condensation of chromatin associated with entry into M-phase. APCFZR1 activity would therefore maintain prophase I arrest by degrading cyclin B1 and in so doing prevent a rise in CDK1 kinase activity. However, with respect to oocytes we currently do not understand how Fzr1 activity is fine tuned, such that cyclin B1 is maintained at sufficient levels to allow oocytes to resume meiosis but not at too high a level to resume meiosis prematurely. Here we describe how intracellular partitioning would explain how this regulation is achieved. Using an oocyte cDNA library, generated from prophase I oocytes in Swiss Quackenbush mice, we found only one splice variant, Fzr1α/Cdh1α, of the single-copy mammalian fzr1 gene, in contrast to various splice variants we found in other tissues. This gene product contained a predicted nuclear localization signal. By immunofluorescence we confirmed this nuclear location, both in isolated oocytes from mice and in oocytes contained in antral follicles from ovarian sections. In fact all the degradation machinery appeared enhanced in the nucleus, as observed by immunofluorescence of APC6, an essential APC subunit and PSMD11, an essential 26S proteasome subunit. In contrast, cyclin B1 was predominantly cytoplasmic. In all systems studied it has been demonstrated that cyclin B1 shuttles between the cytoplasm and nucleus, with nuclear localization occurring just before nuclear envelope breakdown. We reasoned therefore that the nuclear localization of the APCFZR1 and 26S proteasome would aid in maintaining low nuclear levels of cyclin B1. To test this idea further we used two cyclin B mutants, coupled to GPF, which differed in their intracellular location. F146A-cyclin B1, which accumulates in the nucleus due to a single point mutation in the nuclear export signal, and 5A-cyclin B1 in which 5 S/T residues, phosphorylated on cyclin B1 entry into the nucleus, are alanine substituted. We found that nuclear accumulation of cyclin B1 was necessary in order for it to promote meiotic resumption because over-expression of F146A-cyclin B1 accelerated entry into meiosis, whereas 5A-cyclin B1 did not despite the same level of expression. However, in oocytes maintained in prophase I arrested through addition of milrinone, rates of F146A-cyclin B1 degradation were 5 fold (6% vs 32%) that of 5A-cyclin B1. We conclude that in oocytes, an increase in the nuclear-cytoplasmic ratio of cyclin B1 is an essential step in meiotic resumption. During prophase I the constant shuttling of cyclin B1 between nucleus and cytoplasm presents the possibility of precocious meiotic entry, but we argue that this is physiologically prevented by nuclear APCFZR1 activity. Research supported by the NHMRC (Grant 569202) to KTJ. (platform)

A Centrosomal Cdc20-APC Pathway Controls Dendrite Morphogenesis in Postmitotic Neurons

The ubiquitin ligase anaphase-promoting complex (APC) recruits the coactivator Cdc20 to drive mitosis in cycling cells. However, the nonmitotic functions of Cdc20-APC have remained unexplored. We report that Cdc20-APC plays an essential role in dendrite morphogenesis in postmitotic neurons. Knockdown of Cdc20 in cerebellar slices and in postnatal rats in vivo profoundly impairs the formation of granule neuron dendrite arbors in the cerebellar cortex. Remarkably, Cdc20 is enriched at the centrosome in neurons, and the centrosomal localization is critical for Cdc20-dependent dendrite development. We also find that the centrosome-associated protein histone deacetylase 6 (HDAC6) promotes the polyubiquitination of Cdc20, stimulates the activity of centrosomal Cdc20-APC, and drives the differentiation of dendrites. These findings define a postmitotic function for Cdc20-APC in the morphogenesis of dendrites in the mammalian brain. The identification of a centrosomal Cdc20-APC ubiquitin signaling pathway holds important implications for diverse biological processes, including neuronal connectivity and plasticity.

Different phosphorylation states of the anaphase promoting complex in response to antimitotic drugs: A quantitative proteomic analysis

The anaphase promoting complex (APC) controls the degradation of proteins during exit from mitosis and entry into S-phase. The activity of the APC is regulated by phosphorylation during mitosis. Because the phosphorylation pattern provides insights into the complexity of regulation of the APC, we studied in detail the phosphorylation patterns at a single mitotic state of arrest generated by various antimitotic drugs. We examined the phosphorylation patterns of the APC in HeLa S3 cells after they were arrested in prometaphase with taxol, nocodazole, vincristine, or monastrol. There were 71 phosphorylation sites on nine of the APC subunits. Despite the common state of arrest, the various antimitotic drug treatments resulted in differences in the phosphorylation patterns and phosphorylation stoichiometries. The relative phosphorylation stoichiometries were determined by using a method adapted from the isotope-free quantitation of the extent of modification (iQEM). We could show that during drug arrest the phosphorylation state of the APC changes, indicating that the mitotic arrest is not a static condition. We discuss these findings in terms of the variable efficacy of antimitotic drugs in cancer chemotherapy.

Tetratricopeptide repeats in the anaphase‐promoting complex provide multiple activator binding sites

The anaphase‐promoting complex (APC) is a 13‐subunit ubiquitin ligase that initiates the final events of mitosis. Substrates are recruited to the APC by an activator protein (Cdc20 or Cdh1). We tested the possibility that activator binding to the APC depends on three related APC subunits (Cdc27, Cdc16, and Cdc23) that contain multiple tetratricopeptide repeats (TPRs): repeated 34 amino acid sequences that often fold into hydrophobic grooves used for protein‐protein interactions. Each subunit contains 9–10 TPRs, which together may form a large array of TPRs on the APC surface. To assess the function of this array, we mutated residues predicted to lie in the TPR grooves of Cdc27, Cdc16, and Cdc23. Mutation of single residues in the grooves of Cdc27 or Cdc23 reduced the ability of the activators Cdh1 and Cdc20 to bind and activate purified APC. Subunit binding and APC integrity were unaffected. The mutations suppressed the lethality of activator overexpression in yeast. We propose that the presence of multiple activator binding sites may explain how the APC can make specific yet flexible interactions with a wide variety of substrates. Supported by NIH GM53270 and an NSF fellowship.

Accumulation of Substrates of the Anaphase-Promoting Complex (APC) during Human Cytomegalovirus Infection Is Associated with the Phosphorylation of Cdh1 and the Dissociation and Relocalization of APC Subunits

Cell cycle dysregulation upon human cytomegalovirus (HCMV) infection of human fibroblasts is associated with the inactivation of the anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase, and accumulation of its substrates. Here, we have further elucidated the mechanism(s) by which HCMV-induced inactivation of the APC occurs. Our results show that Cdh1 accumulates in a phosphorylated form that may prevent its association with and activation of the APC. The accumulation of Cdh1, but not its phosphorylation, appears to be cyclin-dependent kinase dependent. The lack of an association of exogenously added Cdh1 with the APC from infected cells indicates that the core APC also may be impaired. This is further supported by an examination of the localization and composition of the APC. Coimmunoprecipitation studies show that both Cdh1 and the subunit APC1 become dissociated from the complex. In addition, immunofluorescence analysis demonstrates that as the infection progresses, several subunits redistribute to the cytoplasm, while APC1 remains nuclear. Dissociation of the core complex itself would account for not only the observed inactivity but also its inability to bind to Cdh1. Taken together, these results illustrate that HCMV has adopted multiple mechanisms to inactivate the APC, which underscores its importance for a productive infection.

Cell adhesion induces p27Kip1-associated cell-cycle arrest through down-regulation of the SCFSkp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

AbstractMounting evidence suggests that dynamic interactions between a tumor and its microenvironment play a critical role in tumor development, cell-cycle progression, and response to therapy. In this study, we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stromal cells resulted in a reversible G1 arrest associated with elevated p27Kip1 and p21 (WAF1) proteins. The adhesion-mediated p27Kip1 and p21 increases were posttranslationally regulated via the down-regulation of Skp2, a subunit of SCFSkp2 ubiquitin ligase. Overexpression of Skp2 in MCL decreased p27Kip1, whereas inhibition of Skp2 by siRNA increased p27Kip1 and p21 levels. Furthermore, we found cell adhesion up-regulated Cdh1 (an activating subunit of anaphase-promoting complex [APC] ubiquitin ligase), and reduction of Cdh1 by siRNA induced Skp2 accumulation and hence p27Kip1 degradation, thus implicating Cdh1 as an upstream effector of the Skp2/p27Kip1 signaling pathway. Overall, this report, for the first time, demonstrates that cell-cell contact controls the tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHLs. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches for modifying tumor cell growth and response to therapy.

HER4 D-Box Sequences Regulate Mitotic Progression and Degradation of the Nuclear HER4 Cleavage Product s80HER4

Heregulin-mediated activation of HER4 initiates receptor cleavage (releasing an 80-kDa HER4 intracellular domain, s80(HER4), containing nuclear localization sequences) and results in G(2)-M delay by unknown signaling mechanisms. We report herein that s80(HER4) contains a functional cyclin B-like sequence known as a D-box, which targets proteins for degradation by anaphase-promoting complex (APC)/cyclosome, a multisubunit ubiquitin ligase. s80(HER4) ubiquitination and proteasomal degradation occurred during mitosis but not during S phase. Inhibition of an APC subunit (APC2) using short interfering RNA knockdown impaired s80(HER4) degradation. Mutation of the s80(HER4) D-box sequence stabilized s80(HER4) during mitosis, and s80(HER4)-dependent growth inhibition via G(2)-M delay was significantly greater with the D-box mutant. Polyomavirus middle T antigen-transformed HC11 cells expressing s80(HER4) resulted in smaller, less proliferative, more differentiated tumors in vivo than those expressing kinase-dead s80(HER4) or the empty vector. Cells expressing s80(HER4) with a disrupted D-box did not form tumors, instead forming differentiated ductal structures. These results suggest that cell cycle-dependent degradation of s80(HER4) limits its growth-inhibitory action, and stabilization of s80(HER4) enhances tumor suppression, thus providing a link between HER4-mediated growth inhibition and cell cycle control.

Differential display identifies genes in chinese hamster ovary cells sensitive to elevated ammonium

Ammonium is a toxic waste product that has been reported to negatively inhibit cell growth and recombinant glycosylation in Chinese hamster ovary (CHO) cells; however, the effect of this toxicity on intracellular gene expression has received only limited investigation. We used a differential display method to identify genes in CHO cells that were affected by ammonium stress. Eight genes whose mRNA levels significantly changed in response to elevated ammonium were isolated and identified. Five of the genes were identified as having lower expression under the ammonium stress, whereas three genes were identified as having higher expression. Sequence homology with other mammalian organisms was used to attribute function to these newly identified genes. The identified ammonium-sensitive genes were grouped into three broad functional groups: cellular processes, energy metabolism, and genetic-information processing. The three cellular process-related genes had lower expression (anaphase-promoting complex subunit 5, eukaryotic initiation factor 5A II, KIAA1091 protein). The two energy-related genes had higher expression under ammonium stress (adenosine triphosphate synthase subunit C and mitofusin 1). Both of the genetic information-processing genes (endoplasmic reticulum [ER]-resident protein ERdj5 and structure-specific recognition protein 1) had lower expression under the ammonium stress, whereas the 26S proteasome subunit adenosine triphosphatase 3 gene had higher expression. These preliminary results indicate that ammonium stress lowers expression of genes controlling cell cycle, protein folding, and quality and raises genes that control energy metabolism and degradation. Our findings demonstrate the usefulness of mRNA differential-display techniques for the detection of CHO cell genes affected by ammonium stress.

Components of the Spindle Assembly Checkpoint Regulate the Anaphase-Promoting Complex During Meiosis inCaenorhabditis elegans

Temperature-sensitive mutations in subunits of the Caenorhabditis elegans anaphase-promoting complex (APC) arrest at metaphase of meiosis I at the restrictive temperature. Embryos depleted of the APC co-activator FZY-1 by RNAi also arrest at this stage. To identify regulators and potential substrates of the APC, we performed a genetic suppressor screen with a weak allele of the APC subunit MAT-3/CDC23/APC8, whose defects are specific to meiosis. Twenty-seven suppressors that resulted in embryonic viability and larval development at the restrictive temperature were isolated. We have identified the molecular lesions in 18 of these suppressors, which correspond to five genes. In addition to a single intragenic suppressor, we found mutations in the APC co-activator fzy-1 and in three spindle assembly checkpoint genes, mdf-1, mdf-2, and mdf-3/san-1, orthologs of Mad1, Mad2, and Mad3, respectively. Reduction-of-function alleles of mdf-2 and mdf-3 suppress APC mutants and exhibit pleiotropic phenotypes in an otherwise wild-type background. Analysis of a single separation-of-function allele of mdf-1 suggests that MDF-1 has a dual role during development. These studies provide evidence that components of the spindle assembly checkpoint may regulate the metaphase-to-anaphase transition in the absence of spindle damage during C. elegans meiosis.

Cell Adhesion Induces p27Kip1-Associated Cell-Cycle Arrest through Down-Regulating SCFSkp2 Ubiquitin Ligase Pathway in Mantle Cell and Other Non-Hodgkin's B-Cell Lymphomas.

Mounting evidence now suggests that dynamic interactions between the tumor cell and its local and systemic microenvironment play a critical role in tumor development and that all of the clinical properties of a tumor, including cell cycle progression and response to therapy, depend heavily on the tumor stroma. However, the molecular mechanisms of the interaction between stroma and tumor cells are not well defined. In this study we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stroma cell (HS-5) resulted in a reversible G1 arrest associated with elevated p27Kip1 protein level. The adhesion mediated p27Kip1 increase was posttranslational and regulated through down-regulating Skp2, a substrate-recognition subunit of SCFSkp2 ubiquitin ligase. Overexpression of wild-type Skp2 in MCL decreases p27Kip1, whereas inhibition of Skp2 expression by siRNA increases p27Kip1 level. Furthermore, we found that cell adhesion up-regulated Cdh1, an activating subunit of anaphase-promoting complex (APC) ubiquitin ligase, and reducing Cdh1 by siRNA induced Skp2 accumulation and hence p27Kip1 degradation, implicating Cdh1 as an upstream effector of Skp2/p27Kip1 signaling pathway. Overall, this is a first report demonstrating that the bone marrow stroma controls tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHL. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches modifying tumor cell growth and response to therapy.

Differential mitotic checkpoint protein requirements in somatic and germ cells

Cdc20 (cell division cycle 20) and Cdh1 are the activating subunits of APC (anaphase-promoting complex), an E3-ubiquitin ligase that drives cells into anaphase by inducing degradation of cyclin B and the anaphase inhibitor securin. To prevent chromosome missegregation due to early degradation of cyclin B and securin, mitotic checkpoint protein complexes consisting of BubR1, Bub3 and Mad2 bind to and inhibit APC(Cdc20) until all chromosomes are properly attached to the mitotic spindle and aligned in the metaphase plate. The nuclear transport factors Rae1 and Nup98, which convert into mitotic checkpoint proteins in M-phase, further prevent chromosome missegregation by assembling into a complex with APC(Cdh1) and delaying APC(Cdh1)-mediated ubiquitination of securin. Disruption of Mad2, BubR1, Bub3 or Rae1 in mice results in substantial aneuploidy in somatic tissues, but whether these genes are equally important for accurate chromosome segregation during meiosis has not yet been established. To address this issue, we generated cohorts of male mice in which Mad2, BubR1, Bub3, Rae1 and Nup98 were disrupted either individually or in combination. We tested the fertility of these mice and performed chromosome counts on secondary spermatocytes. We found that male fertility and accurate chromosome segregation during spermatogenesis are highly dependent on BubR1, but not Mad2, Bub3, Rae1 and Nup98. Our results suggest that the mechanisms ensuring accurate chromosome segregation differ between mitotic and meiotic cells.

Genome of Crocodilepox Virus

Here, we present the genome sequence, with analysis, of a poxvirus infecting Nile crocodiles (Crocodylus niloticus) (crocodilepox virus; CRV). The genome is 190,054 bp (62% G+C) and predicted to contain 173 genes encoding proteins of 53 to 1,941 amino acids. The central genomic region contains genes conserved and generally colinear with those of other chordopoxviruses (ChPVs). CRV is distinct, as the terminal 33-kbp (left) and 13-kbp (right) genomic regions are largely CRV specific, containing 48 unique genes which lack similarity to other poxvirus genes. Notably, CRV also contains 14 unique genes which disrupt ChPV gene colinearity within the central genomic region, including 7 genes encoding GyrB-like ATPase domains similar to those in cellular type IIA DNA topoisomerases, suggestive of novel ATP-dependent functions. The presence of 10 CRV proteins with similarity to components of cellular multisubunit E3 ubiquitin-protein ligase complexes, including 9 proteins containing F-box motifs and F-box-associated regions and a homologue of cellular anaphase-promoting complex subunit 11 (Apc11), suggests that modification of host ubiquitination pathways may be significant for CRV-host cell interaction. CRV encodes a novel complement of proteins potentially involved in DNA replication, including a NAD(+)-dependent DNA ligase and a protein with similarity to both vaccinia virus F16L and prokaryotic serine site-specific resolvase-invertases. CRV lacks genes encoding proteins for nucleotide metabolism. CRV shares notable genomic similarities with molluscum contagiosum virus, including genes found only in these two viruses. Phylogenetic analysis indicates that CRV is quite distinct from other ChPVs, representing a new genus within the subfamily Chordopoxvirinae, and it lacks recognizable homologues of most ChPV genes involved in virulence and host range, including those involving interferon response, intracellular signaling, and host immune response modulation. These data reveal the unique nature of CRV and suggest mechanisms of virus-reptile host interaction.

Arabidopsis Anaphase-Promoting Complexes: Multiple Activators and Wide Range of Substrates Might Keep APC Perpetually Busy

The anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase, is anessential regulator of the cell cycle from metaphase until S phase in yeast and metazoans.APC mediates degradation of numerous cell cycle-related proteins, including mitoticcyclins and its activation and substrate-specificity are determined by two adaptor proteins,Cdc20 and Cdh1. Plants have multiple APC activators and the Cdh1-type proteins, inaddition, are represented by two subclasses, known as Ccs52A and Ccs52B. TheArabidopsis genome contains five cdc20 genes as well as ccs52A1, ccs52A2 and ccs52B. InSchizosaccharomyces pombe, expression of the three Atccs52 genes elicited distinctphenotypes supporting non-redundant function of the AtCcs52 proteins. Consistent withthese activities, the AtCcs52 proteins were able to bind both to the yeast and theArabidopsis APCs. In synchronized Arabidopsis cell cultures the cdc20 transcripts werepresent from early G2 until the M-phase exit, ccs52B from G2/M to M while ccs52A1 andccs52A2 were from late M until early G2, suggesting consecutive action of these APCactivators in the plant cell cycle. The AtCcs52 proteins interacted with different subsets ofmitotic cyclins, in accordance with their expression profiles, either in free- or CDK-boundforms. Expression of most APC subunits was constitutive, whereas cdc27a and cdc27b,corresponding to two forms of apc3, and ubc19 and ubc20 encoding E2-C type ubiquitinconjugatingenzymes displayed differences in their cell cycle regulation. These dataindicate the existence of numerous APCCdc20/Ccs52/Cdc27 forms in Arabidopsis, which inconjunction with different E2 enzymes might have distinct or complementary functions atdistinct stages of the cell cycle.

The APC Subunit Doc1 Promotes Recognition of the Substrate Destruction Box

Accurate chromosome segregation during mitosis requires the coordinated destruction of the mitotic regulators securin and cyclins. The anaphase-promoting complex (APC) is a multisubunit ubiquitin-protein ligase that catalyzes the polyubiquitination of these and other proteins and thereby promotes their destruction. How the APC recognizes its substrates is not well understood. In mitosis, the APC activator Cdc20 binds to the APC and is thought to recruit substrates by interacting with a conserved target protein motif called the destruction box. A related protein, called Cdh1, performs a similar function during G1. Recent evidence, however, suggests that the core APC subunit Doc1 also contributes to substrate recognition. To better understand the mechanism by which Doc1 promotes substrate binding to the APC, we generated a series of point mutations in Doc1 and analyzed their effects on the processivity of substrate ubiquitination. Mutations that reduce Doc1 function fall into two classes that define spatially and functionally distinct regions of the protein. One region, which includes the carboxy terminus, anchors Doc1 to the APC but does not influence substrate recognition. The other region, located on the opposite face of Doc1, is required for Doc1 to enhance substrate binding to the APC. Importantly, stimulation of binding by Doc1 also requires that the substrate contain an intact destruction box. Cells carrying DOC1 mutations that eliminate substrate recognition delay in mitosis with high levels of APC substrates. Doc1 contributes to recognition of the substrate destruction box by the APC. This function of Doc1 is necessary for efficient substrate proteolysis in vivo.

Leukemic B Cells from CD38 Positive but Not CD38 Negative B-CLL Patients Express Heightened Levels of Cell Cycle Related Genes.

Within the past five years studies published from this laboratory and others have identified or confirmed CD38 as a marker for aggressive disease in B cell chronic lymphocytic leukemia (B-CLL). To learn more about the possible role of CD38 in this disease, we performed gene expression profiling (GEP; Affymetrix U133A platform) on primary leukemic B cells isolated from CD38 positive and negative patients. We and others have previously shown that the leukemic compartment within some patients consists of both CD38neg and CD38pos CLL B cells. To optimize identification of differentially expressed genes associated with CD38 expression or lack thereof, we restricted our gene profiling to patient cells that were unimodally negative for CD38 and those that expressed unimodally high levels of CD38. In both groups of patients, CD19 positive cells were purified using magnetic bead cell sorting on a Miltenyi AutoMacs prior to isolation of total RNA. To determine genes that appear to be differentially expressed in this set of experiments, we used a recently developed model-based algorithm that uses the normalized probe-level data as well as information about the underlying experimental design. Reassuringly, the gene expression profiles within the CD38pos versus the CD38neg cohort maintain congruency within each cohort when analyzing the gene expression data set as a whole, as well as within categories such as adhesion, signaling, and transcriptional molecules. Using this analytical approach, several genes emerged as interesting candidates for exploration into the nature of the CD38pos association with disease aggressiveness. Interestingly, although peripheral blood CLL B cells are overwhelmingly in the resting, G0, stage of the cell cycle, several cell cycle related genes were expressed at higher levels in CD38pos patient leukemic cells but not in CD38neg leukemic patients. Moreover, these genes encompass protein families that function at different stages throughout the cell cycle. These genes include: anaphase promoting complex subunit 5 (APC5); cell division cycle (cdc) family members; a checkpoint protein CHFR; the microtubule and actin associated protein, MACF1; the mitotic spindle checkpoint protein, MAD1L1; the DNA replication regulator, MCM7; STAG3, a meiotic regulator of chromosome segregation; and several activators of the ubiquitin pathway, including UBE2N, a known regulator of DNA repair enzymes (p-values of <0.01, <0.01, <0.01, 0.01, 0.02, <0.01, 0.01, and 0.01, respectively). We have begun to confirm these findings at the protein translation level. Through immunoblotting, preliminary data show that 3 out of 5 CD38pos and 0 out of 5 CD38neg patients' leukemic cells express detectable levels of APC5, suggesting that CD38pos cells, or at least a subpopulation of cells, express APC5. Taken together, these gene and protein expression data suggest that at least a subpopulation of the leukemic cells from CD38pos CLL patients have more recently completed the cell cycle and maintain expression of specific cell cycle related genes. Alternatively, it is possible that this subpopulation is better prepared to re-enter the cell cycle if it encounters proliferative stimuli in other microenvironments.