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Biomedical subjects

D O Morgan

Publications and source records attributed to D O Morgan.

At least 37 records · Page 2Linked to original sources
PubMed1998

In vivo analysis of the stability and fitness of variants recovered from foot-and-mouth disease virus quasispecies.

We have analysed the ability to infect pigs of two foot-and-mouth disease virus (FMDV) variants isolated at low frequencies from virus populations (quasispecies) generated in pigs on infection with a parental virus, C-S8c1. A monoclonal antibody-resistant mutant (MARM21), and a variant isolated at early times post-infection (S-3T1), each exhibiting a unique amino acid substitution in VP1, were able to cause disease in pigs, both by direct inoculation or by contact transmission. The symptoms developed were similar to those produced by C-S8c1 or the related virus C-S15c1. The VP1 sequence of viral RNA directly recovered from lesions of infected animals confirmed the stability of the variant genotypes. Pigs infected with S-3T1 consistently showed an advance of 12 to 24 h in the emergence of fever and lesions when compared to animals infected with C-S8c1 or the remaining variants, an observation consistent with its early isolation. The ability of FMDV variants to compete in vivo with C-S8c1 was investigated in co-infection experiments. Analysis of the proportion of each of the competitors in lesions of co-infected pigs revealed that none of the variants was completely overgrown by the parent. However, co-infection with C-S8c1 and MARM21 resulted in lesions in which C-S8c1 was predominant, indicating a selective disadvantage of this variant in swine. In contrast, lesions from swine co-infected with C-S8c1 and S-3T1 contained similar proportions of the two viruses. These results document fitness variations in vivo among components of the mutant spectrum of FMDV quasispecies.

Animals↗
PubMed1998

Cak1 is required for Kin28 phosphorylation and activation in vivo.

Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1 mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.

Animals↗
PubMed1997

The HIV transactivator TAT binds to the CDK-activating kinase and activates the phosphorylation of the carboxy-terminal domain of RNA polymerase II.

The human immunodeficiency virus encodes the transcriptional transactivator Tat, which binds to the transactivation response (TAR) RNA stem-loop in the viral long terminal repeat (LTR) and increases rates of elongation rather than initiation of transcription by RNA polymerase II (Pol II). In this study, we demonstrate that Tat binds directly to the cyclin-dependent kinase 7 (CDK7), which leads to productive interactions between Tat and the CDK-activating kinase (CAK) complex and between Tat and TFIIH. Tat activates the phosphorylation of the carboxy-terminal domain (CTD) of Pol II by CAK in vitro. The ability of CAK to phosphorylate the CTD can be inhibited specifically by a CDK7 pseudosubstrate peptide that also inhibits transcriptional activation by Tat in vitro and in vivo. We conclude that the phosphorylation of the CTD by CAK is essential for Tat transactivation. Our data identify a cellular protein that interacts with the activation domain of Tat, demonstrate that this interaction is critical for the function of Tat, and provide a mechanism by which Tat increases the processivity of Pol II.

Animals↗
PubMed1997

Neutralization antigenic sites on type Asia-1 foot-and-mouth disease virus defined by monoclonal antibody-resistant variants.

Seven neutralizing monoclonal antibodies (nMAbs) produced against serotype Asia-1 foot-and-mouth disease virus (FMDV) were used to select neutralization-resistant variants. Seven single and six multiple antibody-resistant variants were selected to identify neutralization antigenic sites on FMDV Asia-1. The variants no longer reacted with nMAbs which were used to select them when tested by microneutralization test (MNT), radioimmunoassay (RIA) and agar gel immunodiffusion (AGID) assay. Based on the binding and neutralization patterns of the variants, the nMAbs could be divided into discrete groups indicating the presence of three independent antigenic sites with evidence for occurrence of possibly a fourth site on the virus surface. Site 1 was present on 140S, 12Sps and VP1 and thus was conformation-independent. Sites 2 and 3 were restricted to the intact virion (140S) and thus were more conformation-dependent. Site 4 present on 140S virions and 12S protein subunits was less conformation-dependent. The site 3 nMAbs neutralized the infectivity of all the ten different Asia-1 virus isolates tested indicating that this site is conserved in Asia-1 virus serotype. Both cross-neutralization of different Asia-1 viruses with the nMAbs and cross-inhibition assays between MAbs demonstrated that the nMAbs recognized at least six different epitopes on Asia-1 virus.

Animals↗
PubMed1997

Cyclin-dependent kinases: engines, clocks, and microprocessors.

Cyclin-dependent kinases (Cdks) play a well-established role in the regulation of the eukaryotic cell division cycle and have also been implicated in the control of gene transcription and other processes. Cdk activity is governed by a complex network of regulatory subunits and phosphorylation events whose precise effects on Cdk conformation have been revealed by recent crystallographic studies. In the cell, these regulatory mechanisms generate an interlinked series of Cdk oscillators that trigger the events of cell division.

Animals↗
PubMed1996

A cyclin-dependent kinase-activating kinase (CAK) in budding yeast unrelated to vertebrate CAK.

Progress through the cell cycle is governed by the cyclin-dependent kinases (CDKs), the activation of which requires phosphorylation by the CDK-activating kinase (CAK). In vertebrates, CAK is a trimeric enzyme containing CDK7, cyclin H, and MAT1. CAK from the budding yeast Saccharomyces cerevisiae was identified as an unusual 44-kilodalton protein kinase, Cak1, that is only distantly related to CDKs. Cak1 accounted for most CAK activity in yeast cell lysates, and its activity was constant throughout the cell cycle. The CAK1 gene was essential for cell viability. Thus, the major CAK in S. cerevisiae is distinct from the vertebrate enzyme, suggesting that budding yeast and vertebrates may have evolved different mechanisms of CDK activation.

Amino Acid Sequence↗
PubMed1996

The dynamics of cyclin dependent kinase structure.

In the past year, several new crystal structures have provided exciting insights into the conformational changes underlying the regulation of cyclin-dependent kinases. We now understand the structural basis of many of the mechanisms by which cyclin-dependent kinases are regulated, including activation by cyclin binding and phosphorylation, inhibition by the inhibitor p27, and binding by the CKS proteins.

Cell Division↗
PubMed1996

Three-dimensional structure of human cyclin H, a positive regulator of the CDK-activating kinase.

Cyclin-dependent kinases (CDKs), which play a key role in cell cycle control, are activated by the CDK activating kinase (CAK), which activates cyclin-bound CDKs by phosphorylation at a specific threonine residue. Vertebrate CAK contains two key components: a kinase subunit with homology to its substrate CDKs and a regulatory subunit with homology to cyclins. We have determined the X-ray crystal structure of the regulatory subunit of CAK, cyclin H, at 2.6 A resolution. Cyclin H contains two alpha-helical core domains with a fold similar to that of cyclin A, a regulatory subunit of CAK substrate CDK2, and of TFIIB, a transcription factor. Outside of the core domains, the N- and C-terminal regions of the three structures are completely different. The conformational differences between cyclin H and A structures may reflect functional differences between the two cyclins.

Amino Acid Sequence↗
PubMed1996

Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells.

The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X-irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.

CDC2 Protein Kinase↗
PubMed1995

Alternative mechanisms of CAK assembly require an assembly factor or an activating kinase.

We have cloned a mouse cDNA that encodes p36, a novel subunit of the CDK-activating kinase (CAK). p36 contains a C3HC4 zinc-binding domain or RING factor and is associated both with a TFIIH-bound form of CAK and with a free trimeric form. p36 promotes the assembly of CDK7 and cyclin H in vitro, stabilizing the transient CDK7-cyclin H complex. Stabilization and activation of CAK by p36 is independent of the phosphorylation state of T170, the conserved activating residue of CDK7. Assembly of active CDK7-cyclin H dimers can also occur through an alternative p36-independent pathway that requires phosphorylation of T170 by a CAK-activating kinase, or CAKAK. Thus, CDK7-cyclin H complex formation can be achieved by multiple mechanisms.

Amino Acid Sequence↗
PubMed1995

c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism.

We have explored the role of the tyrosine kinase c-Src in cellular adhesion. Fibroblasts derived from src-/- mice (src-/- fibroblasts) exhibit a reduced rate of spreading on fibronectin. These defect is rescued by expression of wild-type chicken c-Src. Analyses of mutants suggest that c-Src increases the rate of cell spreading in src-/- fibroblasts through a kinase-independent mechanism requiring both the SH3 and SH2 domains. To further address the role of c-Src in adhesion, we examined the activity and subcellular distribution of c-Src during the adhesion of fibroblasts on fibronectin. We observed a transient increase in the specific kinase activity of c-Src accompanied by the partial dephosphorylation of the negative regulatory site Y527. Activation of c-Src is followed by its redistribution to newly formed focal adhesions. These results suggest that the enzymatic activity and subcellular distribution of c-Src are coordinately regulated during cellular adhesion and that c-Src can affect adhesion by a kinase-independent mechanism.

Animals↗
PubMed1995

Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins.

Studies of the temperature-sensitive cdc37-1 mutant of Saccharomyces cerevisiae suggest that Cdc37 is required for passage through the G1 phase of the cell cycle, but its precise function is not known. We have investigated the role of Cdc37 in the regulation of the cyclin-dependent protein kinase Cdc28. We find that G1 arrest in the cdc37-1 mutant is accompanied by a decrease in the Cdc28 activity associated with the G1 cyclin Cln2. This defect appears to be caused by a decrease in the binding of Cdc28 and Cln2. cdc37-1 mutants also exhibit a defect in the binding and activation of Cdc28 by the mitotic cyclin Clb2. Thus Cdc37 may be a regulator that is required for the association of Cdc28 with multiple cyclins.

Base Sequence↗
PubMed1995

Cdk-activating kinase complex is a component of human transcription factor TFIIH.

Transcription factor IIH (TFIIH) contains a kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). Here we report the identification of the Cdk-activating kinase (Cak) complex (Cdk7 and cyclin H) as a component of TFIIH after extensive purification of TFIIH by chromatography. We find that affinity-purified antibodies directed against cyclin H inhibit TFIIH-dependent transcription and that both cyclin H and Cdk7 antibodies inhibit phosphorylation of the CTD of the largest subunit of the RNAPII in the preinitiation complex. Cak is present in at least two distinct complexes, TFIIH and a smaller complex that is unable to phosphorylate RNAPII in the preinitiation complex. Both Cak complexes, as well as recombinant Cak, phosphorylate a CTD peptide. Finally, TFIIH was shown to phosphorylate both Cdc2 and Cdk2, suggesting that there could be a link between transcription and the cell cycle machinery.

Amino Acid Sequence↗
PubMed1995

Principles of CDK regulation.

As key regulators of the cell cycle, the cyclin-dependent kinases must be tightly regulated by extra- and intracellular signals. The activity of cyclin-dependent kinases is controlled by four highly conserved biochemical mechanisms, forming a web of regulatory pathways unmatched in its elegance and intricacy.

Animals↗
PubMed1995

Multiple modes of ligand recognition: crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors, olomoucine and isopentenyladenine.

Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(delta 2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties.

Adenine↗