From molecules to slides: EPStoPICTTM and epsConverterTM for the Macintosh.
Explore the source record for details and available documents.
Computer Graphics↗
Biomedical subjects
T Hunt
Publications and source records attributed to T Hunt.
Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.
The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone.
Animals↗
The crystal structure of cyclin A.
BACKGROUND: Eukaryotic cell cycle progression is regulated by cyclin dependent protein kinases (CDKs) whose activity is regulated by association with cyclins and by reversible phosphorylation. Cyclins also determine the subcellular location and substrate specificity of CDKs. Cyclins exhibit diverse sequences but all share homology over a region of approximately 100 amino acids, termed the cyclin box. From the determination of the structure of cyclin A, together with results from biochemical and genetic analyses, we can identify which parts of the cyclin molecular may contribute to cyclin A structure and function. RESULTS: We have solved the crystal structure, at 2.0 A resolution, of an active recombinant fragment of bovine cyclin A, cyclin A-3, corresponding to residues 171-432 of human cyclin A. The cyclin box has an alpha-helical fold comprising five alpha helices. This fold is repeated in the C-terminal region, although this region shares negligible sequence similarity with the cyclin box. CONCLUSIONS: Analysis of residues that are conserved throughout the A, B, and E cyclins identifies two exposed clusters of residues, one of which has recently been shown to be involved in the association with human CDK2. The second cluster may identify another site of cyclin A-protein interaction. Comparison of the structure of the unbound cyclin with the structure of cyclin A complexed with CDK2 reveals that cyclin A does not undergo any significant conformational changes on complex formation. Threading analysis shows that the cyclin-box fold is consistent with the sequences of the transcription factor TFIIB and other functionally related proteins. The structural results indicate a role for the cyclin-box fold both as a template for the cyclin family and as a generalised adaptor molecule in the regulation of transcription.
Algorithms↗
Identification of a developmental timer regulating the stability of embryonic cyclin A and a new somatic A-type cyclin at gastrulation.
We have identified a second Xenopus cyclin A, called cyclin A2. Cyclin A2 is a 46.6-kD protein that shows a greater homology to human cyclin A than to the previously identified Xenopus cyclin A1. It is present throughout embryonic development (up to stage 46 at least) and is found in adult tissues as well as in Xenopus tissue culture cell lines. In contrast, cyclin A1 is present in eggs and early embryos but cannot be detected in late embryos or in tissue culture cells. We have found that the maternally stored pools of mRNAs encoding both of these cyclin A proteins are stable until the onset of gastrulation and then are degraded abruptly. At this time, new transcription replaces cyclin A2 mRNA. Interestingly, we have also observed a dramatic change in the stability of the cyclin A proteins at this time. Prior to the onset of gastrulation, cyclin A1 protein is stable during interphase of the cell cycle. At gastrulation, however, both A1 and A2 proteins turn over rapidly during interphase of the cell cycle. Together, these results indicate that developmental programs controlling cyclin A protein and mRNA stability are activated at gastrulation. We have shown that this program is independent of new transcription beginning at the mid-blastula transition. Furthermore, treatment of early stage embryos with cycloheximide demonstrates that activation of this degradative program is independent of cell division and translation. Collectively, our observations suggest that a previously uncharacterized timing mechanism activates new degradative pathways at the onset of gastrulation, which could play an essential role in releasing cells from maternal programming.
Amino Acid Sequence↗
Cyclin A and cyclin B dissociate from p34cdc2 with half-times of 4 and 15 h, respectively, regardless of the phase of the cell cycle.
The exchange rate of cyclin A and cyclin B between endogenous p34cdc2 and bacterially expressed GST-cdc2 was measured in concentrated Xenopus egg extracts. The half-lives of the cyclin A.p34cdc2 and the cyclin B.p34cdc2 complexes are estimated as 4 and 15 h, respectively. There is no significant difference in these affinities when they are measured in mitosis, interphase, or during transition between these two states. The tight association between cyclin and p34cdc2 in the cell cycle may be significant for mechanisms of cyclin destruction.
Amino Acid Sequence↗
Cip1 blocks the initiation of DNA replication in Xenopus extracts by inhibition of cyclin-dependent kinases.
BACKGROUND: Cip1 is a 21 kD protein that interacts with and inhibits cyclin-dependent kinases (cdks). Expression of Cip1 is induced by the tumour suppressor p53, and tumour cells have greatly reduced levels of Cip1. As cdks are required for normal progression through the cell cycle, their inhibition by Cip1 may mediate the ability of p53 to block cell proliferation. Cip1 has also been shown to inhibit the DNA polymerase delta auxiliary factor PCNA (proliferating cell nuclear antigen), which is required for replication-fork elongation, and this could be an alternative mechanism by which p53-induced Cip1 blocks cell proliferation. RESULTS: We have investigated the effect of Cip1 protein on chromosomal DNA replication, using cell-free extracts of Xenopus eggs that initiate and complete chromosome replication under normal cell-cycle control. Cip1 protein strongly inhibited an early stage of DNA replication in this system, and this inhibition was not complemented by extracts that had been affinity-depleted of cdks. In contrast, Cip1 did not inhibit the elongation of replication forks that had accumulated in the presence of aphidicolin. Cip1 inhibition of DNA replication was fully rescued by addition of cyclins A or E, but not cyclin B, cdk2 or PCNA. CONCLUSIONS: Our results suggest that Cip1 specifically blocks the initiation of DNA replication by inhibition of a cyclin-dependent kinase (cdk2), but has no major effect on the elongation of preassembled replication forks. The ability of cyclin A or cyclin E to rescue the Cip1 inhibition suggests that these cyclins may play a direct role in the initiation of replication in the Xenopus system.
Animals↗
A brain-specific activator of cyclin-dependent kinase 5.
Phosphorylation of the neurofilament proteins of high and medium relative molecular mass, as well as of the Alzheimer's tau protein, is thought to be catalysed by a protein kinase with Cdc2-like substrate specificity. We have purified a novel Cdc2-like kinase from bovine brain capable of phosphorylating both the neurofilament proteins and tau. The purified enzyme is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a novel regulatory subunit, p25 (ref. 8). When overexpressed and purified from Escherichia coli, p25 can activate Cdk5 in vitro. Unlike Cdk5, which is ubiquitously expressed in human tissue, the p25 transcript is expressed only in brain. A full-length complementary DNA clone showed that p25 is a truncated form of a larger protein precursor, p35, which seems to be the predominant form of the protein in crude brain extract. Cdk5/p35 is the first example of a Cdc2-like kinase with neuronal function.
Amino Acid Sequence↗
A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins.
MAPK-activated protein kinase-2 (MAPKAP kinase-2) is activated in vitro by the p42 and p44 isoforms of MAPK (p42/p44MAPK). In several cell lines, however, MAPKAP kinase-2 is activated by sodium arsenite, heat shock, or osmotic stress and not by agonists that activate p42/p44MAPK. We have identified a MAPK-like enzyme that acts as a MAPKAP kinase-2 reactivating kinase (RK). RK is recognized by an antiserum raised against a Xenopus MAPK (Mpk2), which is most similar to HOG1 from S. cerevisiae. We also identified a RK kinase (RKK) on the basis of its ability to activate either RK or a GST-Mpk2 fusion protein. The RKK, RK, and MAPKAP kinase-2 constitute a new stress-activated signal transduction pathway in vertebrates that is distinct from the classical MAPK cascade.
Amino Acid Sequence↗
The 3'-untranslated regions of c-mos and cyclin mRNAs stimulate translation by regulating cytoplasmic polyadenylation.
Early in the development of many animals, before transcription begins, any change in the pattern of protein synthesis is attributable to a change in the translational activity or stability of an mRNA in the egg. As a result, translational control is critical for a variety of developmental decisions, including axis formation in Drosophila and sex determination in Caenorhabditis elegans. Previous work demonstrated that increases in poly(A) length can activate translation, whereas removal of poly(A) can prevent it. In this report we focus on the control of c-mos and cyclin A1, B1, and B2 mRNAs during meiotic maturation and after fertilization of frog eggs. We show that addition and removal of poly(A) from these mRNAs is extensively regulated: The time at which each mRNA receives or loses poly(A), as well as the number of adenosines it gains or loses, differ substantially. Signals in the 3'-untranslated region (UTR) of each mRNA are sufficient to reconstitute both the temporal and quantitative control of poly(A) addition: Chimeric mRNAs in which a luciferase-coding region is joined to the 3' UTRs of cyclin A1, cyclin B1, or c-mos mRNA, receive poly(A) of the same length and at the same time as do the endogenous mRNAs. Moreover, each 3' UTR also regulates translation of the chimeric mRNAs, determining when and how much translation of the luciferase reporter is stimulated during maturation. The magnitude of stimulation in luciferase activity varies from 5- to 100-fold, depending on the 3' UTR. Translational stimulation by each 3' UTR requires poly(A) lengthening, as it is prevented by mutations that prevent that process. These results suggest that the 3' UTRs of cyclin and c-mos mRNAs control not only whether or not an mRNA is turned on during maturation, but when that activation occurs and to what extent. Translational control of c-mos mRNA, which may be achieved through regulation of poly(A) length, may be critical in the activation of maturation, and in the onset of cleavage divisions. Our findings, as well as those of others, suggest that even quite complex patterns of translational activation in the early embryo can be attained through the differential control of a common mechanism.
Animals↗
Destruction of Xenopus cyclins A and B2, but not B1, requires binding to p34cdc2.
The specific and rapid destruction of cyclins A and B during mitosis is their most remarkable property. A short peptide motif of approximately 10 amino acids near the N-terminus, known as the destruction box, is absolutely required for programmed proteolysis. In this paper we show that although the destruction box is necessary for the degradation of cyclin A, it is not sufficient. Mutant versions of cyclin A that cannot form complexes with p34cdc2 are stable, which we interpret to mean that this cyclin must bind to p34cdc2 in order to undergo programmed proteolysis. Thus, N-terminal fragments of cyclin A containing little more than the destruction box and its surroundings are indestructible. p34cdc2 binding also appears to be required for the destruction of cyclin B2. In contrast, cyclin B1 does not require p34cdc2 binding for specific proteolysis. The systems for the proteolysis of cyclins A, B1 and B2 thus appear to show important differences in the way they recognize their substrates.
Amino Acid Sequence↗
Assay and purification of Fv fragments in fermenter cultures: design and evaluation of generic binding reagents.
Fv fragments whose genes have been cloned using common PCR primers carry identical peptide motifs at their termini. We have raised antibodies against the C-terminal motif of the VH chain GQGTTVTVSS and evaluated their utility as reagents for the assay and purification of Fvs in the fermenter culture. Three different Fvs were included in the investigation. We found that the motif was exposed and available for capture when Fv fragments were blotted onto nitrocellulose paper or adsorbed directly onto microtiter plates. In contrast, the motif was either partially or totally obscured when the Fv was complexed with immobilised antigen or when free in solution. This reactivity profile enabled us to develop a general-purpose assay for Fv protein, but not a general-purpose assay for monitoring active Fv. The apparent inaccessibility of the C-terminus of VH conflicts with currently held views on the three-dimensional structure of these molecules.
Amino Acid Sequence↗
Reversible immunoprecipitation using histidine- or glutathione S-transferase-tagged staphylococcal protein A.
We have constructed, expressed, and purified hexahistidine- and glutathione S-transferase (GST)-tagged Staphylococcal protein A. The histidine-tagged protein A bound efficiently to iminodiacetic acid (IDA)-Sepharose loaded with Zn2+, and the GST-protein A was efficiently retained by glutathione-Sepharose. Both recombinant forms of protein A can be used in the normal way to harvest immune complexes with IgG. Both forms of protein A can be released from the Sepharose matrix by mild procedures. The his6-protein A:antibody:antigen complexes can be released from the matrix with EDTA, and immunoprecipitates bound to GST-protein A can be released either by elution with glutathione or by digestion with thrombin. We tested this method with immunoprecipitates of the p40MO15 protein kinase, and found that they retained their ability to phosphorylate p33cdk2 after elution from the affinity matrices.
Amino Acid Sequence↗
Invasion of the cabbage patch (should biochemists learn the Krebs cycle or the cell cycle?)
Explore the source record for details and available documents.
Biochemistry↗
Radiographic diagnosis of ankle fractures: are three views necessary?
One hundred and twenty-three sets of emergency room ankle x-rays (anteroposterior lateral and mortise) were retrospectively reviewed to determine whether all three views were necessary to diagnose the presence of an ankle fracture. Four physicians (two orthopaedic surgeons, one musculoskeletal radiologist, and one emergency room physician) reviewed all randomly ordered sets of films twice--once with all three views and once with only the lateral and mortise views. The overall accuracy of two views was within the 95% expected threshold of accuracy using three views. The lateral and mortise views alone appear sufficient for ankle fracture diagnosis, and imply a substantial decrease in radiation and cost savings to the patient.
Ankle Injuries↗
Cell cycle regulation of the p34cdc2/p33cdk2-activating kinase p40MO15.
A key component of Cdc2/Cdk2-activating kinase (CAK) is p40MO15, a protein kinase subunit that phosphorylates the T161/T160 residues of p34cdc2/p33cdk2. The level and activity of p40MO15 were essentially constant during cleavage of fertilised Xenopus eggs and in growing mouse 3T3 cells, but serum starvation of these cells reduced both the level and activity of p40MO15. Although the level and activity of endogenous p40MO15 did not vary in the cell cycle, we found that bacterially expressed p40MO15 was activated more rapidly by M-phase cell extracts than by interphase cell extracts. Bacterially expressed p40MO15 was phosphorylated mainly on serine 170 (a p34cdc2 phosphorylation site) by mitotic cell extracts, but mutation of S170 to alanine did not affect the activation of p40MO15, whereas mutation of T176 (the equivalent site to T161/T160 in p34cdc2/p33cdk2) abolished the activation of P40MO15. These studies suggest that the level and activity of p40MO15 is probably not a major determinant of p34cdc2/p33cdk2 activity in the cell cycle, and that the activation of p40MO15 may require phosphorylation on T176.
3T3 Cells↗
Similarities between somatic cells overexpressing the mos oncogene and oocytes during meiotic interphase.
The mos protooncogene encodes a serine/threonine kinase and is a key regulator of oocyte meiotic maturation. After acute infection of Swiss 3T3 cells with virus containing the v-mos oncogene, cells expressing high levels of v-Mos round up and detach from the monolayer (floating cells), while cells that remain attached express 10-fold lower levels of v-Mos and are transformed. The floating cells are growth arrested with their chromosomes partially condensed in the absence of histone H1 kinase activity, while mitogen-activated protein kinase activity is very high. Collectively, these properties are similar to properties observed in maturing oocytes between meiosis I and II. In v-mos-transformed cell populations, mitogen-activated protein kinase activity is also elevated, correlating with the degree of morphological transformation and the level of Mos expression. Moreover, phosphoprotein modifications specific for M are found in both the floating cells and in v-mos-transformed cells, regardless of their cell cycle stage. One explanation for both morphological transformation and the phenotypes of the floating cells is that Mos imposes a meiotic program on different stages of the somatic cell cycle. The extent of this meiotic phenotype is proportional to the level of v-Mos expression. These results suggest that both morphological transformation and the phenotypes of the floating cells induced by Mos in Swiss 3T3 cells are related to its normal activities during oocyte maturation.
3T3 Cells↗
Cell cycle. Dams and sluices.
Explore the source record for details and available documents.
Cell Cycle↗
The protein kinase mos activates MAP kinase kinase in vitro and stimulates the MAP kinase pathway in mammalian somatic cells in vivo.
The mos protooncogene encodes a serine/threonine protein kinase that is only expressed at significant levels in germ cells. Recombinant malE-mos protein (Xenopus mos protooncogene fused in frame to the maltose binding protein of E. coli) activates MAP kinase in cell-free extracts prepared from Xenopus oocytes and eggs. Here we show that malE-mos immunoprecipitates from Xenopus extracts phosphorylate and activate MAP kinase kinase in vitro, indicating that mos can function as a MAP kinase kinase kinase. Moreover, ectopic expression of mos in mammalian somatic cells, that lack any endogenous mos protein, triggers the activation of MAP kinase in vivo. These results identify the mos protooncogene as a direct activator of the MAP kinase pathway, with the potential to activate this kinase cascade even in cells where normally there is no expression of mos.
3T3 Cells↗