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Biomedical subjects

J Wade Harper

Publications and source records attributed to J Wade Harper.

At least 37 records · Page 2Linked to original sources
PubMed2005

Identification and functional evaluation of cellular and viral factors involved in the alteration of nuclear architecture during herpes simplex virus 1 infection.

Herpes simplex virus 1 (HSV-1) replicates in the nucleus of host cells and radically alters nuclear architecture as part of its replication process. Replication compartments (RCs) form, and host chromatin is marginalized. Chromatin is later dispersed, and RCs spread past it to reach the nuclear edge. Using a lamin A-green fluorescent protein fusion, we provide direct evidence that the nuclear lamina is disrupted during HSV-1 infection and that the UL31 and UL34 proteins are required for this. We show nuclear expansion from 8 h to 24 h postinfection and place chromatin rearrangement and disruption of the lamina in the context of this global change in nuclear architecture. We show HSV-1-induced disruption of the localization of Cdc14B, a cellular protein and component of a putative nucleoskeleton. We also show that UL31 and UL34 are required for nuclear expansion. Studies with inhibitors of globular actin (G-actin) indicate that G-actin plays an essential role in nuclear expansion and chromatin dispersal but not in lamina alterations induced by HSV-1 infection. From analyses of HSV infections under various conditions, we conclude that nuclear expansion and chromatin dispersal are dispensable for optimal replication, while lamina rearrangement is associated with efficient replication.

Actins↗
PubMed2005

HiNF-P directly links the cyclin E/CDK2/p220NPAT pathway to histone H4 gene regulation at the G1/S phase cell cycle transition.

Genome replication in eukaryotic cells necessitates the stringent coupling of histone biosynthesis with the onset of DNA replication at the G1/S phase transition. A fundamental question is the mechanism that links the restriction (R) point late in G1 with histone gene expression at the onset of S phase. Here we demonstrate that HiNF-P, a transcriptional regulator of replication-dependent histone H4 genes, interacts directly with p220(NPAT), a substrate of cyclin E/CDK2, to coactivate histone genes during S phase. HiNF-P and p220 are targeted to, and colocalize at, subnuclear foci (Cajal bodies) in a cell cycle-dependent manner. Genetic or biochemical disruption of the HiNF-P/p220 interaction compromises histone H4 gene activation at the G1/S phase transition and impedes cell cycle progression. Our results show that HiNF-P and p220 form a critical regulatory module that directly links histone H4 gene expression at the G1/S phase transition to the cyclin E/CDK2 signaling pathway at the R point.

Amino Acid Motifs↗
PubMed2004

Recognition of phosphodegron motifs in human cyclin E by the SCF(Fbw7) ubiquitin ligase.

Turnover of cyclin E is controlled by SCF(Fbw7). Three isoforms of Fbw7 are produced by alternative splicing. Whereas Fbw7alpha and -gamma are nuclear and the beta-isoform is cytoplasmic in 293T cells, all three isoforms induce cyclin E destruction in an in vivo degradation assay. Cyclin E is phosphorylated on Thr(62), Ser(88), Ser(372), Thr(380), and Ser(384) in vivo. To examine the roles of phosphorylation in cyclin E turnover, a series of alanine point mutations in each of these sites were analyzed for Fbw7-driven degradation. As expected, mutation of the previously characterized residue Thr(380) to alanine led to profound defects of cyclin E turnover, and largely abolished association with Fbw7. Mutation of Thr(62) to alanine led to a dramatic reduction in the extent of Thr(380) phosphorylation, suggesting an indirect effect of this mutation on cyclin E turnover. Nevertheless, phosphopeptides centered at Thr(62) associated with Fbw7, and residual binding of cyclin E(T380A) to Fbw7 was abolished upon mutation of Thr(62), suggesting a minor role for this residue in direct association with Fbw7. Mutation of Ser(384) to alanine also rendered cyclin E resistant to degradation by Fbw7, with the largest effects being observed with Fbw7beta. Cyclin E(S384A) associated more weakly with Fbw7alpha and -beta isoforms but was not defective in Thr(380) phosphorylation. Analysis of the localization of cyclin E mutant proteins indicated selective accumulation of cyclin E(S384A) in the nucleus, which may contribute to the inability of cytoplasmic Fbw7beta to promote turnover of this cyclin E mutant protein.

Amino Acid Motifs↗
PubMed2004

Interwoven ubiquitination oscillators and control of cell cycle transitions.

Ubiquitin-mediated proteolysis has emerged as a paramount mechanism for regulating the cell division cycle. Changes in the activities of certain E3 ligases can promote the interconversion of cell cycle states or transitions. Recent studies have revealed how distinct E3 ligases control the activity of other E3 ligases and how the interplay between these degradation machines sets up the timing of cell cycle transitions. For example, during G1, the anaphase-promoting complex in conjunction with Cdh1 (APC(Cdh1)) catalyzes destruction of the S-phase activator Skp2, helping to define the G1 state. In response to poorly defined signals, APC(Cdh1) activity is reduced, allowing accumulation of Skp2 and therefore entry into S phase. In many cases, E3 ligases also function to ubiquitinate proteins that negatively regulate cell cycle transitions. Recent work indicates that cyclin-dependent kinase 2 and Polo kinase collaborate to phosphorylate Wee1, thereby promoting its ubiquitination by SCF(beta-TRCP). Thus, activation of the mitotic transition produces feedback signals that help to turn off the negative upstream pathway to further reenforce the transition.

Animals↗
PubMed2004

Neddylating the guardian; Mdm2 catalyzed conjugation of Nedd8 to p53.

The tumor suppressor and transcriptional regulator p53 is perhaps one of the most regulated proteins in the cell nucleus and is acted upon by a variety of protein kinases, acetylases, ubiqutin ligases and hydrolases, and SUMO-conjugating enzymes. Now new work suggests a role for an additional modification-neddylation-in negative regulation of p53 transcriptional activity.

Catalysis↗
PubMed2004

The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation.

Myc proteins regulate cell growth and division and are implicated in a wide range of human cancers. We show here that Fbw7, a component of the SCF(Fbw7) ubiquitin ligase and a tumor suppressor, promotes proteasome-dependent c-Myc turnover in vivo and c-Myc ubiquitination in vitro. Phosphorylation of c-Myc on threonine-58 (T58) by glycogen synthase kinase 3 regulates the binding of Fbw7 to c-Myc as well as Fbw7-mediated c-Myc degradation and ubiquitination. T58 is the most frequent site of c-myc mutations in lymphoma cells, and our findings suggest that c-Myc activation is one of the key oncogenic consequences of Fbw7 loss in cancer. Because Fbw7 mediates the degradation of cyclin E, Notch, and c-Jun, as well as c-Myc, the loss of Fbw7 is likely to elicit profound effects on cell proliferation during tumorigenesis.

Base Sequence↗
PubMed2004

Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein.

The mammalian F-box protein Fbw7 and its Caenorhabditis elegans counterpart Sel-10 have been implicated in the ubiquitin-mediated turnover of cyclin E as well as the Notch/Lin-12 family of transcriptional activators. Both unregulated Notch and cyclin E promote tumorigenesis, and inactivating mutations in human Fbw7 suggest that it may be a tumor suppressor. To generate an in vivo system to assess the consequences of such unregulated signaling, we generated mice deficient for Fbw7. Fbw7-null mice die around 10.5 days post coitus because of a combination of deficiencies in hematopoietic and vascular development and heart chamber maturation. The absence of Fbw7 results in elevated levels of cyclin E, concurrent with inappropriate DNA replication in placental giant trophoblast cells. Moreover, the levels of both Notch 1 and Notch 4 intracellular domains were elevated, leading to stimulation of downstream transcriptional pathways involving Hes1, Herp1, and Herp2. These data suggest essential functions for Fbw7 in controlling cyclin E and Notch signaling pathways in the mouse.

Animals↗
PubMed2004

Negative regulation of SCFSkp2 ubiquitin ligase by TGF-beta signaling.

TGF-beta is a multifunctional growth factor whose best-known function is to inhibit cell growth and suppress tumor formation. TGF-beta causes cells to accumulate in mid-to-late G1 phase by blocking the transition from G1 to S. It has been shown that TGF-beta inhibits Cdk2-cyclin E kinase activity by promoting the binding of cell cycle inhibitor p27Kip1 to the kinase complexes. Here, we show that TGF-beta treatment leads to stabilization of p27Kip1 during G1 to S transition. We found that TGF-beta negatively regulates components of the SCF complex, which degrades the p27Kip1 during the G1 to S transition, through two distinct mechanisms. Using a pulse-chase analysis, we demonstrated that the stability of Skp2 decreases in the presence of TGF-beta. Destabilization of Skp2 by ubiquitin-mediated proteolysis was also demonstrated that in an in vitro degradation system, using cell extracts prepared from TGF-beta-treated cultured cells. In addition, TGF-beta treatment decreases the levels of Cks1 mRNA. The deficiency of Cks1 in TGF-beta-treated cells likely contributes to the stabilization of p27Kip1 and destabilization of Skp2, because in the absence of Cks1, SCFSkp2 cannot ubiquitinate p27Kip1; instead, self-ubiquitination of Skp2 occurs. Thus, stabilization of the cell cycle inhibitor p27Kip1 and cell growth inhibition in response to TGF-beta occur in part through limiting the threshold of the SCFSkp2 ubiquitin ligase by transcriptional and post-transcriptional mechanisms.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2004

A map of the interactome network of the metazoan C. elegans.

To initiate studies on how protein-protein interaction (or "interactome") networks relate to multicellular functions, we have mapped a large fraction of the Caenorhabditis elegans interactome network. Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from high-throughput, yeast two-hybrid (HT=Y2H) screens. Independent coaffinity purification assays experimentally validated the overall quality of this Y2H data set. Together with already described Y2H interactions and interologs predicted in silico, the current version of the Worm Interactome (WI5) map contains approximately 5500 interactions. Topological and biological features of this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological hypotheses.

Animals↗
PubMed2004

Visualization of a highly organized intranuclear network of filaments in living mammalian cells.

For 30 years, the mammalian cell nucleus has been hypothesized to contain a filamentous framework, the nuclear matrix or karyoskeleton, which regulates nuclear structure and function. However, such an organized network of filaments has never been observed in living cells. Here we show that human Cdc14B phosphatase in living cells tightly associates with long filaments that begin at the nucleolar periphery and extend to the nuclear envelope, frequently making close connections with nuclear pore complexes. We demonstrate that Cdc14B contains a bipartite signal that directs it to the intranuclear filaments, and we also detect a small amount of Cdc14B on interphase and mitotic centrosomes. Furthermore, we show that Cdc14B is critical for the maintenance of proper nuclear structure together with polo-like kinase Plk1. This work provides the first direct evidence for the existence of an intranuclear filamentous framework in living mammalian cells and implicates Cdc14B in the control of mammalian nuclear architecture.

Animals↗
PubMed2004

Cyclin/CDK regulates the nucleocytoplasmic localization of the human papillomavirus E1 DNA helicase.

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.

Active Transport, Cell Nucleus↗
PubMed2004

The Keap1-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase: oxidative stress sensing by a Cul3-Keap1 ligase.

The Nrf2 transcription factor promotes survival following cellular insults that trigger oxidative damage. Nrf2 activity is opposed by the BTB/POZ domain protein Keap1. Keap1 is proposed to regulate Nrf2 activity strictly through its capacity to inhibit Nrf2 nuclear import. Recent work suggests that inhibition of Nrf2 may also depend upon ubiquitin-mediated proteolysis. To address the contribution of Keap1-dependent sequestration versus Nrf2 proteolysis, we identified the E3 ligase that regulates Nrf2 ubiquitination. We demonstrate that Keap1 is not solely a cytosolic anchor; rather, Keap1 is an adaptor that bridges Nrf2 to Cul3. We demonstrate that Cul3-Keap1 complexes regulate Nrf2 polyubiquitination both in vitro and in vivo. Inhibition of either Keap1 or Cul3 increases Nrf2 nuclear accumulation, leading to promiscuous activation of Nrf2-dependent gene expression. Our data demonstrate that Keap1 restrains Nrf2 activity via its capacity to target Nrf2 to a cytoplasmic Cul3-based E3 ligase and suggest a model in which Keap1 coordinately regulates both Nrf2 accumulation and access to target genes.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Cyclin F disruption compromises placental development and affects normal cell cycle execution.

Human cyclin F was originally isolated as a cDNA capable of suppressing the temperature sensitivity of a Saccharomyces cerevisiae cdc4-1 mutant. Its tightly regulated expression and high conservation in the evolutionary progression from amphibians to mammals suggest that it coordinates the timing of a critical cell cycle event. The fact that it contains an F box and can form an SCF (Skp1-Cul1/Cdc53-F-box) complex in vivo further suggests that it may also function in proteolysis. To investigate the role of cyclin F in vivo, we generated mice deficient for cyclin F and conditionally deficient mice as well as mouse embryonic fibroblasts (MEFs) conditionally deficient for cyclin F. Heterozygous animals are normal and fertile, but CycF(-/-) animals, with a myriad of developmental anomalies due in large part to failures in yolk sac and chorioallantoic placentation, die around embryonic day 10.5. Tissue-specific deletion of cyclin F revealed that it was not required for the development and function of a number of different embryonic and adult tissues. In contrast, MEFs lacking cyclin F, while viable, do exhibit cell cycle defects, including reduced population-doubling time and a delay in cell cycle reentry from quiescence, indicating that cyclin F plays a role in cell cycle regulation.

Animals↗
PubMed2003

SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase.

Eukaryotic cells respond to DNA damage and stalled replication forks by activating protein kinase-mediated signaling pathways that promote cell cycle arrest and DNA repair. A central target of the cell cycle arrest program is the Cdc25A protein phosphatase. Cdc25A is required for S-phase entry and dephosphorylates tyrosine-15 phosphorylated Cdk1 (Cdc2) and Cdk2, positive regulators of cell division. Cdc25A is unstable during S-phase and is degraded through the ubiquitin-proteasome pathway, but its turnover is enhanced in response to DNA damage. Although basal and DNA-damage-induced turnover depends on the ATM-Chk2 and ATR-Chk1 pathways, how these kinases engage the ubiquitin ligase machinery is unknown. Here, we demonstrate a requirement for SCFbeta-TRCP in Cdc25A turnover during an unperturbed cell cycle and in response to DNA damage. Depletion of beta-TRCP stabilizes Cdc25A, leading to hyperactive Cdk2 activity. SCFbeta-TRCP promotes Chk1-dependent Cdc25A ubiquitination in vitro, and this involves serine 76, a known Chk1 phosphorylation site. However, recognition of Cdc25A by beta-TRCP occurs via a noncanonical phosphodegron in Cdc25A containing phosphoserine 79 and phosphoserine 82, sites that are not targeted by Chk1. These data indicate that Cdc25A turnover is more complex than previously appreciated and suggest roles for an additional kinase(s) in Chk1-dependent Cdc25A turnover.

Amino Acid Motifs↗
PubMed2003

An oriented peptide array library (OPAL) strategy to study protein-protein interactions.

One of the major questions in signal transduction is how the specificities of protein-protein interactions determine the assembly of distinct signaling complexes in response to stimuli. Several peptide library methods have been developed and widely used to study protein-protein interactions. These approaches primarily rely on peptide or DNA sequencing to identify the peptide or consensus motif for binding and may prove too costly or difficult to accommodate high throughput applications. We report here an oriented peptide array library (OPAL) approach that should facilitate high throughput proteomic analysis of protein-protein interactions. OPAL integrates the principles of both the oriented peptide libraries and array technologies. Hundreds of pools of oriented peptide libraries are synthesized as amino acid scan arrays. We demonstrate that these arrays can be used to map the specificities of a variety of interactions, including antibodies, protein domains such Src homology 2 domains, and protein kinases.

Animals↗
PubMed2003

BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3.

Programmed destruction of regulatory proteins through the ubiquitin-proteasome system is a widely used mechanism for controlling signalling pathways. Cullins are proteins that function as scaffolds for modular ubiquitin ligases typified by the SCF (Skp1-Cul1-F-box) complex. The substrate selectivity of these E3 ligases is dictated by a specificity module that binds cullins. In the SCF complex, this module is composed of Skp1, which binds directly to Cul1, and a member of the F-box family of proteins. F-box proteins bind Skp1 through the F-box motif, and substrates by means of carboxy-terminal protein interaction domains. Similarly, Cul2 and Cul5 interact with BC-box-containing specificity factors through the Skp1-like protein elongin C. Cul3 is required for embryonic development in mammals and Caenorhabditis elegans but its specificity module is unknown. Here we report the identification of a large family of BTB-domain proteins as substrate-specific adaptors for C. elegans CUL-3. Biochemical studies using the BTB protein MEL-26 and its genetic target MEI-1 (refs 12, 13) indicate that BTB proteins merge the functional properties of Skp1 and F-box proteins into a single polypeptide.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Exit from exit: resetting the cell cycle through Amn1 inhibition of G protein signaling.

In S. cerevisiae cells undergoing anaphase, a ras-related GTPase, Tem1, is located on the spindle pole body that enters the daughter cell and activates a signal transduction pathway, MEN, to allow mitotic exit. MEN activation must be reversed after mitotic exit to reset the cell cycle in G1. We find that daughter cells activate an Antagonist of MEN pathway (AMEN) in part through induction of the Amn1 protein that binds directly to Tem1 and prevents its association with its target kinase Cdc15. Failure of Amn1 function results in defects of both the spindle assembly and nuclear orientation checkpoints and delays turning off Cdc14 in G1. Thus, Amn1 is part of a daughter-specific switch that helps cells exit from mitotic exit and reset the cell cycle.

Binding Sites↗