PubMed Health⌕ Search

Biomedical subjects

J Wade Harper

Publications and source records attributed to J Wade Harper.

53 records · Page 3Linked to original sources
PubMed2003

Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition.

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC-coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2003

Therapeutic anti-cancer targets upstream of the proteasome.

Polyubiquitination of a protein is generally the first step in its degradation. This article discusses how altered protein destruction pathways impact the cell cycle and allow for abnormal cell proliferation, and explores how this process can be utilized in anticancer therapy. There are several levels of possible therapeutic intervention in ubiquitin-dependent proteolysis pathways upstream of the proteasome. In principle, targeting specific components of the ubiquitin system may offer an opportunity to develop selective drugs. However, the fact that general proteasome inhibitors have been demonstrated to be effective in cancer therapy suggests that other ubiquitin components that are common to many destruction pathways may also be clinically useful. We will, therefore, evaluate both the specific, rate-limiting enzymes and a number of general, nonselective enzymes as targets for anticancer therapy. Potential nonselective therapeutic strategies that are under investigation in a variety of human cancers include the identification and inhibition of individual F-box proteins, such as Skp2, and the inhibition of the ubiquitin ligases such as the SCF family, Mdm2, and Efp. A general pathway under investigation is the cullin neddylation and deneddylation system, with promising enzymatic targets such as csn5 and Rpn11.

Breast Neoplasms↗
PubMed2003

Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase.

The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the >70 human F box proteins allowing the recognition of a wide range of substrates. The F box protein beta-TrCP1 recognizes the doubly phosphorylated DpSGphiXpS destruction motif, present in beta-catenin and IkappaB, and directs the SCF(beta-TrCP1) to ubiquitinate these proteins at specific lysines. The 3.0 A structure of a beta-TrCP1-Skp1-beta-catenin complex reveals the basis of substrate recognition by the beta-TrCP1 WD40 domain. The structure, together with the previous SCF(Skp2) structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant beta-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection.

Amino Acid Motifs↗
PubMed2003

A license to kill: transcriptional activation and enhanced turnover of Myc by the SCF(kp2) ubiquitin ligase.

Understanding the mechanisms through which the abundance and activity of the Myc oncoprotein is regulated has been a major preoccupation of the cancer and transcription communities. New work, published in the May issue of Molecular Cell, reveals that ubiquitination of Myc by the oncogenic SCF(Skp2) complex not only promotes Myc turnover but is also required for Myc transcriptional activation, suggesting that Myc activity is "licensed" by ubiquitination.

Humans↗
PubMed2003

Deafening cycle.

Explore the source record for details and available documents.

Animals↗
PubMed2003

The cyclin E/Cdk2 substrate and Cajal body component p220(NPAT) activates histone transcription through a novel LisH-like domain.

p220(NPAT) is a substrate of cyclin E/Cdk2 that localizes in nuclear organelles called Cajal bodies in a cell cycle-regulated manner. In normal diploid fibroblasts, p220 is concentrated in two Cajal bodies tethered to histone gene clusters at chromosome 6p21 during G(1), S, and G(2) phases and two additional Cajal bodies tethered to histone genes at 1q21 during S, and G(2) phases. Overexpression of p220 in U2OS cells can promote the G(1)/S transition and can also promote transcription from histone H2B and H4 luciferase reporter constructs. How p220 expression induces these activities and whether the two activities are related are unknown. In this study, we developed a "lox-scanning" mutagenesis approach to identify functional domains in p220. We identified two distinct functional regions of p220. The C-terminal half of the protein contains multiple elements that are required for its ability to induce S phase in transfected cells. In contrast, sequences at the N terminus appear to be critical for activation of histone H4 and H2B reporter constructs. We identified an approximately 30-amino-acid motif at the N terminus of p220 that has the characteristics of a LisH motif. LisH motifs are found in a large number of proteins in the database but are of unknown function. Point mutations in conserved residues in the LisH motif of p220 block histone H4 transcriptional activity without affecting localization in Cajal bodies or phosphorylation on Cdk2 phosphorylation sites. These studies indicate that the ability of p220 to promote S phase is independent of its ability to promote histone H4 transcription and suggests that p220 may link cyclin E/Cdk2 to multiple independent downstream functions.

Amino Acid Motifs↗
PubMed2003

Identification of HiNF-P, a key activator of cell cycle-controlled histone H4 genes at the onset of S phase.

At the G(1)/S phase cell cycle transition, multiple histone genes are expressed to ensure that newly synthesized DNA is immediately packaged as chromatin. Here we have purified and functionally characterized the critical transcription factor HiNF-P, which is required for E2F-independent activation of the histone H4 multigene family. Using chromatin immunoprecipitation analysis and ligation-mediated PCR-assisted genomic sequencing, we show that HiNF-P interacts with conserved H4 cell cycle regulatory sequences in vivo. Antisense inhibition of HiNF-P reduces endogenous histone H4 gene expression. Furthermore, we find that HiNF-P utilizes NPAT/p220, a substrate of the cyclin E/cyclin-dependent kinase 2 (CDK2) kinase complex, as a key coactivator to enhance histone H4 gene transcription. The biological role of HiNF-P is reflected by impeded cell cycle progression into S phase upon antisense-mediated reduction of HiNF-P levels. Our results establish that HiNF-P is the ultimate link in a linear signaling pathway that is initiated with the growth factor-dependent induction of cyclin E/CDK2 kinase activity at the restriction point and culminates in the activation of histone H4 genes through HiNF-P at the G(1)/S phase transition.

Amino Acid Sequence↗
PubMed2003

The cyclin E/Cdk2 substrate p220(NPAT) is required for S-phase entry, histone gene expression, and Cajal body maintenance in human somatic cells.

Cyclin E/Cdk2, a central regulator of the G1/S transition, coordinates multiple cell cycle events, including DNA replication, centrosome duplication, and activation of the E2F transcriptional program. Recent studies suggest a role for cyclin E/Cdk2 in activation of histone transcription during S phase via the Cajal body-associated protein p220NPAT, and in addition, p220 can promote S-phase entry independently of histone transcriptional activation when overexpressed. Here we have examined the requirement for p220 in histone transcription, cell cycle progression, and Cajal body function through analysis of human somatic HCT116 cells engineered to contain a conditional p220 allele. p220 is required for proliferation of HCT116 cells, as assessed after expression of Cre recombinase in p220(flox/-) cells. This defect was due to an inability of these cells to transit from G0/G1 into S phase, and cell cycle arrest occurred in the presence of elevated Cdk2 kinase activity. Expression of human papillomavirus E7, but not E6, eliminated cell cycle arrest in response to p220 depletion. Optimal expression of all four core histone genes required p220, as did optimal transcription of a histone H4 promoter-luciferase construct. Basal histone H4 expression in G0/G1, although p220 dependent, occurs in the absence of detectable phosphorylation of p220 on Cdk2 sites. Cells lacking p220 displayed defects in the localization of the Cajal body component p80coilin as cells progressed from G0 to S phase in response to mitogenic signals. These finding indicate that p220 is an essential downstream component of the cyclin E/Cdk2 signaling pathway and functions to coordinate multiple elements of the G1/S transition.

CDC2-CDC28 Kinases↗
PubMed2002

Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.

SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold.

Amino Acid Motifs↗
PubMed2002

Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit.

The anaphase-promoting complex (APC) is a multi-subunit E3 protein ubiquitin ligase that is responsible for the metaphase to anaphase transition and the exit from mitosis. One of the subunits of the APC that is required for its ubiquitination activity is Doc1/Apc10, a protein composed of a Doc1 homology domain that has been identified in a number of diverse putative E3 ubiquitin ligases. Here, we present the crystal structure of Saccharomyces cerevisiae Doc1/Apc10 at 2.2A resolution. The Doc1 homology domain forms a beta-sandwich structure that is related in architecture to the galactose-binding domain of galactose oxidase, the coagulation factor C2 domain and a domain of XRCC1. Residues that are invariant amongst Doc1/Apc10 sequences, including a temperature-sensitive mitotic arrest mutant, map to a beta-sheet region of the molecule, whose counterpart in galactose oxidase, the coagulation factor C2 domains and XRCC1, mediate bio-molecular interactions. This finding suggests the identification of the functionally important and conserved region of Doc1/Apc10 and, since invariant residues of Doc1/Apc10 colocalise with conserved residues of other Doc1 homology domains, we propose that the Doc1 homology domains perform common ubiquitination functions in the APC and other E3 ubiquitin ligases.

Amino Acid Sequence↗
PubMed2002

A phosphorylation-driven ubiquitination switch for cell-cycle control.

Cellular changes in state can be dictated by complex all-or-nothing switches built from ultrasensitive protein kinase cascades, positive-feedback loops and other mechanisms. Recent work has established that phosphorylation-driven protein destruction through the SCF ubiquitin-ligase pathway can also occur in a switch-like manner. In this context, multiple phosphorylation events are used to set a threshold for substrate targeting, thereby providing a framework for understanding the inter-relationship between protein phosphorylation and ubiquitin-mediated proteolysis.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2002

RING finger specificity in SCF-driven protein destruction.

SCF ubiquitin ligases contain an E3 core composed of Skp1, Cul1, a member of the Rbx1/Roc1 family of RING finger proteins, and a modular F box protein that functions in substrate targeting. Work published in this issue of Developmental Cell indicates that distinct Rbx1/Roc1 family members are used to control ubiquitination of distinct targets, suggesting that the RING finger subunit may contribute to ubiquitination specificity.

Animals↗
PubMed2002

Coupling of DNA synthesis and histone synthesis in S phase independent of cyclin/cdk2 activity.

DNA and histone synthesis are both triggered at the beginning of S phase by cyclin/cdk2 activity. Previous studies showed that inhibition of DNA synthesis with hydroxyurea or cytosine arabinoside (AraC) triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. Here we show that ectopic expression of HIRA, the likely human ortholog of two cell cycle-regulated repressors of histone gene transcription in yeast (Hir1p and Hir2p), represses transcription of histones and that this, in turn, triggers a concerted block of DNA synthesis. Thus, in mammalian cells sustained DNA synthesis and histone synthesis are mutually dependent on each other during S phase. Although cyclin/cdk2 activity drives activation of both DNA and histone synthesis at the G1/S transition of cycling cells, concerted repression of DNA or histone synthesis in response to inhibition of either one of these is not accompanied by prolonged inhibition of cyclin A/cdk2 or E/cdk2 activity. Therefore, during S phase coupling of DNA and histone synthesis occurs, at least in part, through a mechanism that is independent of cyclin/cdk2 activity. Coupling of DNA and histone synthesis in S phase presumably contributes to the prompt and orderly assembly of newly replicated DNA into chromatin.

Antigens, CD19↗
PubMed2002

Reversal of growth suppression by p107 via direct phosphorylation by cyclin D1/cyclin-dependent kinase 4.

p107 functions to control cell division and development through interaction with members of the E2F family of transcription factors. p107 is phosphorylated in a cell cycle-regulated manner, and its phosphorylation leads to its release from E2F. Although it is known that p107 physically associates with E- and A-type cyclin/cyclin-dependent kinase 2 (Cdk2) complexes through a cyclin-binding RXL motif located in the spacer domain, the mechanisms underlying p107 inactivation via phosphorylation remain poorly defined. Recent genetic evidence indicates a requirement for cyclin D1/Cdk4 complexes in p107 inactivation. In this work, we provide direct biochemical evidence for the involvement of cyclin D1/Cdk4 in the inactivation of p107's growth-suppressive function. While coexpression of cyclin D1/Cdk4 can reverse the cell cycle arrest properties of p107 in Saos-2 cells, we find that p107 in which the Lys-Arg-Arg-Leu sequence of the RXL motif is replaced by four alanine residues is largely refractory to inactivation by cyclin D/Cdk4, indicating a role for this motif in p107 inactivation without a requirement for its tight interaction with cyclin D1/Cdk4. We identified four phosphorylation sites in p107 (Thr-369, Ser-640, Ser-964, and Ser-975) that are efficiently phosphorylated by Cdk4 but not by Cdk2 in vitro and are also phosphorylated in tissue culture cells. Growth suppression by p107 containing nonphosphorylatable residues in these four sites is not reversed by coexpression of cyclin D1/Cdk4. In model p107 spacer region peptides, phosphorylation of S640 by cyclin D1/Cdk4 is strictly dependent upon an intact RXL motif, but phosphorylation of this site in the absence of an RXL motif can be partially restored by replacement of S643 by arginine. This suggests that one role for the RXL motif is to facilitate phosphorylation of nonconsensus Cdk substrates. Taken together, these data indicate that p107 is inactivated by cyclin D1/Cdk4 via direct phosphorylation and that the RXL motif of p107 plays a role in its inactivation by Cdk4 in the absence of stable binding.

Amino Acid Motifs↗
PubMed2002

Elevated Skp2 protein expression in human prostate cancer: association with loss of the cyclin-dependent kinase inhibitor p27 and PTEN and with reduced recurrence-free survival.

The F-box protein Skp2 (Fbl1) is a positive regulator of G1-S transition and promotes ubiquitin-mediated proteolysis of the cyclin-dependent kinase inhibitor p27. Its overexpression has been implicated in cell transformation and oncogenesis in both in vitro and in vivo models. In this study, we investigated its role in human prostate cancer progression. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 622 radical prostatectomy specimens, 74 prostatic intraepithelial neoplasm specimens, as well as in 4 normal prostate organ donors assembled into tissue microarrays. We found that both luminal and basal epithelial cells in normal prostate had very low Skp2 levels, but Skp2 levels and labeling frequency increased dramatically in both premalignant lesions of prostatic intraepithelial neoplasm (P = 0.0252) and in prostate cancer (P = 0.0037). The Skp2 labeling frequency in cancer was positively correlated with preoperative serum prostate-specific antigen level (P = 0.0499) and Gleason score (P = 0.0002), whereas the Skp2 index was positively correlated with extraprostatic extension (P = 0.0454), clinical stage (P = 0.0170), as well as Gleason score (P = 0.0002). Kaplan-Meier analysis revealed that a higher Skp2 labeling index (>10) was a significant predictor of shorter biochemical recurrence-free survival time after radical prostatectomy (P < 0.0363, log-rank test). An inverse correlation of Skp2 was observed with both its biochemical target p27 expression in prostate cancer (P = 0.0003) and with its putative negative regulator, the PTEN tumor suppressor protein (P = 0.0444). These data suggest that induction of Skp2 may be causally linked with decreased levels of p27 in prostate cancer and implicate PTEN in the regulation of Skp2 expression in vivo, as previous tissue culture experiments have suggested.

Cell Cycle Proteins↗