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Biomedical subjects

T Hunt

Publications and source records attributed to T Hunt.

At least 91 records · Page 5Linked to original sources
PubMed1993

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified.

ATP-Binding Cassette Transporters↗
PubMed1993

The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2.

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.

Adenosine Triphosphate↗
PubMed1993

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

CDC2 Protein Kinase↗
PubMed1993

Simian virus 40 large T antigen associates with cyclin A and p33cdk2.

In this paper we provide evidence that a fraction of large T antigen of simian virus 40 (SV40) interacts with cyclin A and p33cdk2 in both virus-infected and stably transformed cells. Immunoprecipitates of SV40 large T antigen from SV40-infected or SV40 large-T-antigen-transformed cells contain cyclin A, p33cdk2, and histone H1 kinase activity. Conversely, immunoprecipitates of cyclin A from these cells contain SV40 large T antigen. In this respect, SV40 large T antigen has properties similar to those of the E1A oncogene of adenoviruses and the E7 oncogene of human papillomaviruses.

Animals↗
PubMed1993

HPV16 E7 protein associates with the protein kinase p33CDK2 and cyclin A.

E7 is the major transforming protein of human papillomavirus type 16 (HPV16). It has been found to associate with the retinoblastoma protein Rb1. We investigated whether HPV16 E7 protein was associated with other cellular proteins, in particular with those involved in cell cycle control. Immunoprecipitates from CaSki cell extracts with an anti E7 monoclonal antibody contained a histone H1 kinase. Recombinant E7, synthesized in yeast, when mixed with protein extracts from epithelial cells bound histone H1 kinase activity in vitro. The in vivo and the in vitro-formed E7-kinase complex had the same periodicity of activity during the cell cycle, being most active in S and G2/M. Immunoblotting of E7 immunoprecipitates with an antibody raised against the p33CDK2, revealed a 33 kDa protein band not detected by an anti-p34cdc2 antibody, suggesting that the E7-associated kinase activity is due to the p33CDK2. The interaction appears to be via cyclin A, since probing of similar immunoblots showed a 50 kDa band corresponding to cyclin A. The association of E7 with cyclin A appeared to be direct, not involving Rb 1 or other proteins.

Cyclins↗
PubMed1992

Plasmid cDNA-directed protein synthesis in a coupled eukaryotic in vitro transcription-translation system.

A system is described in which transcription of cDNA clones by bacteriophage T7 RNA polymerase is coupled to translation in the micrococcal nuclease treated rabbit reticulocyte lysate in a single reaction of coupled transcription-translation. The monovalent and divalent cation requirements for translation are dominant for optimum expression in this coupled system, so that transcription is relatively inefficient. Nevertheless, the use of appropriate DNA concentrations leads to the synthesis of sufficient RNA to saturate the protein synthesis capacity of the system. The fidelity and efficiency of expression in this coupled system are high, and the degree of purification of the plasmid DNA is relatively uncritical. The system therefore offers very considerable advantages for rapid screening of 'mini-preparations' of cDNA plasmid constructs for retention of the correct reading frame and expression of the desired protein product.

Animals↗
PubMed1992

Bone-inducing agent (BIA) from cultured human Saos-2 osteosarcoma cells.

The Saos-2 line of human osteosarcoma cells was established in culture in 1975. These cells produce a large amount of alkaline phosphatase but little or no matrix in vitro, and are unable to grow when transplanted into athymic mice. We decided to test our local strain of Saos-2 cells for bone-inducing ability in the skeletal muscle of athymic mice by implanting freeze-dried, acetone-defatted cells, with and without a collagen carrier. A bone-inducing activity (BIA) thus was demonstrated in 88% of 90 implants of devitalized Saos-2 cells. In further studies, we have used guanidinium hydrochloride (Gu-HCl) to extract, solubilize, and remove the Saos bone-inducing agent(s) in an active state which when reprecipitated by aqueous dialysis was able to induce ultrastructurally typical endochondrial bone formation in nude mouse muscle in 92% of 48 implants. This preliminary report is offered to alert investigators to the presence of an extractable BIA in Saos-2 cells.

Animals↗
PubMed1992

The nature of cytotoxic drug-induced cell death in murine intestinal crypts.

The nature of cell death in murine small intestinal crypts caused by potentially lethal doses of four classes of cancer chemotherapeutic agents was studied. The drugs used were cytosine arabinoside, vincristine, adriamycin and nitrogen mustard. The compounds readily induced massive cell death in the proliferating compartment of the crypt. In each case, cell death was apparent within an hour, and the incidence of dead cells peaked during the following 4-8 h. By 24 h, little damage was discernible in the crypt systems. Remarkably, dead cells or dead cell fragments were phagocytosed rapidly (within about 1 h) by neighbouring healthy enterocytes. When examined by light microscopy, transmission electron microscopy and scanning electron microscopy, the dead cells showed the characteristic features of having succumbed to an apoptotic mode of cell death without any trace of cell and organelle oedema characteristic of necrosis. The study suggests that cell death by apoptosis operates even when the cells are exposed to severe pathological perturbation and that the phenomenon is not solely a process which operates in response to either physiological stimuli or to mild physical or chemical trauma.

Animals↗
PubMed1992

The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo.

Fertilization of clam oocytes initiates a series of cell divisions, of which the first three--meiosis I, meiosis II, and the first mitotic division--are highly synchronous. After fertilization, protein synthesis is required for the successful completion of every division except meiosis I. When protein synthesis is inhibited, entry into meiosis I and the maintenance of M phase for the normal duration of meiosis occur normally, but the chromosomes fail to interact correctly with the spindle in meiosis II metaphase. By contrast, inhibition of protein synthesis immediately after completion of meiosis or mitosis stops cells entering the next mitosis. We describe the behavior of cyclins A and B in relation to these "points of no return." The cyclins are synthesized continuously and are rapidly destroyed shortly before the metaphase-anaphase transition of the mitotic cell cycles, with cyclin A being degraded in advance of cyclin B. Cyclin destruction normally occurs during a 5-min window in mitosis, but in the monopolar mitosis that occurs after parthenogenetic activation of clam oocytes, or when colchicine is added to fertilized eggs about to enter first mitosis, the destruction of cyclin B is strongly delayed, whereas proteolysis of cyclin A is maintained in an activated state for the duration of metaphase arrest. Under either of these abnormal conditions, inhibition of protein synthesis causes a premature return to interphase that correlates with the time when cyclin B disappears.

Animals↗
PubMed1992

Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits.

The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.

Amino Acid Sequence↗
PubMed1992

Cyclin A recruits p33cdk2 to the cellular transcription factor DRTF1.

Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.

Base Sequence↗
PubMed1991

Independent inactivation of MPF and cytostatic factor (Mos) upon fertilization of Xenopus eggs.

In vertebrates, mature eggs are arrested at the second meiotic metaphase by the cytostatic factor (CSF), now known to be the c-mos proto-oncogene product (Mos). Fertilization or egg activation triggers a transient increase in the cytoplasmic free calcium and releases the meiotic arrest by inactivating maturation/mitosis-promoting factor (MPF). CSF or Mos, which is also inactivated by the calcium transient, seems to stabilize MPF in mature eggs and CSF-injected embryos. Thus, it was assumed that CSF inactivation is the primary cause of MPF inactivation on meiotic release. We have directly compared the degradation kinetics of CSF (Mos) and MPF during meiotic release, using the same batch of Xenopus eggs. We report here that, at the molecular level, cyclin subunits of MPF are degraded before Mos is degraded and, at the physiological level, that MPF activity is inactivated before CSF activity during activation of Xenopus eggs. These results, in conjunction with circumstantial evidence, support the novel view that a calcium transient on fertilization induces a CSF-independent pathway for MPF inactivation, whereas CSF inactivation during meiotic release serves only to allow the fertilized egg to enter mitosis.

Animals↗
PubMed1991

Cyclin A and the retinoblastoma gene product complex with a common transcription factor.

The retinoblastoma gene (Rb) product is a negative regulator of cellular proliferation, an effect that could be mediated in part at the transcriptional level through its ability to complex with the sequence-specific transcription factor DRTF1. This interaction is modulated by adenovirus E1a, which sequesters the Rb protein and several other cellular proteins, including cyclin A, a molecule that undergoes cyclical accumulation and destruction during each cell cycle and which is required for cell cycle progression. Cyclin A, which also complexes with DRTF1, facilitates the efficient assembly of the Rb protein into the complex. This suggests a role for cyclin A in regulating transcription and defines a transcription factor through which molecules that regulate the cell cycle in a negative fashion, such as Rb, and in a positive fashion, such as cyclin A, interact. Mutant loss-of-function Rb alleles, which occur in a variety of tumour cells, also fail to complex with E1a and large T antigen. Here we report on a naturally occurring loss-of-function Rb allele encoding a protein that fails to complex with DRTF1. This might explain how mutation in the Rb gene prevents negative growth control.

Animals↗