The structure of CDK4/cyclin D3 has implications for models of CDK activation.

Cyclin-dependent kinase 4 (CDK4)/cyclin D complexes are expressed early in the G(1) phase of the cell cycle and stimulate the expression of genes required for G(1) progression by phosphorylation of the product of the retinoblastoma gene, pRb. To elaborate the molecular pathway of CDK4 activation and substrate selection we have determined the structure of nonphosphorylated CDK4/cyclin D3. This structure of an authentic CDK/cyclin complex shows that cyclin binding may not be sufficient to drive the CDK active site toward an active conformation. Phosphorylated CDK4/cyclin D3 is active as a pRb kinase and is susceptible to inhibition by p27(Kip1). Unlike CDK2/cyclin A, CDK4/cyclin D3 can be inactivated by treatment with lambda-phosphatase, implying that phosphorylated T172 is accessible to a generic phosphatase while bound to a cyclin. Taken together, these results suggest that the structural mechanism of CDK4/cyclin D3 activation differs markedly from that of previously studied CDK/cyclin complexes.

The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases.

Progression through the eukaryotic cell cycle is driven by the orderly activation of cyclin-dependent kinases (CDKs). For activity, CDKs require association with a cyclin and phosphorylation by a separate protein kinase at a conserved threonine residue (T160 in CDK2). Here we present the structure of a complex consisting of phosphorylated CDK2 and cyclin A together with an optimal peptide substrate, HHASPRK. This structure provides an explanation for the specificity of CDK2 towards the proline that follows the phosphorylatable serine of the substrate peptide, and the requirement for the basic residue in the P+3 position of the substrate. We also present the structure of phosphorylated CDK2 plus cyclin A3 in complex with residues 658-668 from the CDK2 substrate p107. These residues include the RXL motif required to target p107 to cyclins. This structure explains the specificity of the RXL motif for cyclins.

The cyclin box fold: protein recognition in cell-cycle and transcription control.

Regulation of both the cell cycle and gene transcription is essential for orderly progression of cell growth and division. Recent results on the structures of two cyclins, cyclin A and cyclin H, and two transcription factor mediator proteins, TFIIB and the A pocket region of the retinoblastoma tumour suppressor protein (Rb), show that they share domains with a strikingly similar alpha-helical topology, despite remote sequence identity.

Conservation of heterochromatin protein 1 function.

Heterochromatin represents a cytologically visible state of heritable gene repression. In the yeast, Schizosaccharomyces pombe, the swi6 gene encodes a heterochromatin protein 1 (HP1)-like chromodomain protein that localizes to heterochromatin domains, including the centromeres, telomeres, and the donor mating-type loci, and is involved in silencing at these loci. We identify here the functional domains of swi6p and demonstrate that the chromodomain from a mammalian HP1-like protein, M31, can functionally replace that of swi6p, showing that chromodomain function is conserved from yeasts to humans. Site-directed mutagenesis, based on a modeled three-dimensional structure of the swi6p chromodomain, shows that the hydrophobic amino acids which lie in the core of the structure are critical for biological function. Gel filtration, gel overlay experiments, and mass spectroscopy show that HP1 proteins can self-associate, and we suggest that it is as oligomers that HP1 proteins are incorporated into heterochromatin complexes that silence gene activity.

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.

Increased glucose uptake capacity of Rous-transformed cells and the relevance of deprivation derepression.

The increased rate of glucose uptake found in cells transformed by Rous sarcoma virus was shown to be enhanced relative to the changes in uptake induced in nontransformed cells by deprivation of glucose (deprivation derepression). Glucose-specific uptake sites were distinguished from glucose-galactose sites in nontransformed cells, and the capacities for glucose uptake and for galactose uptake were increased to about the same extent by the exclusion of glucose from the cell culture medium. Deprivation derepression occurred without a requirement for new RNA or protein synthesis, suggesting that preexisting inactivate uptake sites were activated. Deprivation derepression could be mimicked by the treatment of cells with adenosine triphosphatase activators, and adenosine triphosphate levels were reduced in glucose-deprived cells and in cells treated with adenosine triphosphatase activators. Cells transformed by the Bryan strain of Rous sarcoma virus were unresponsive to addition of high concentrations of glucose, to glucose starvation, or to treatment with adenosine triphosphatase activators, and the relative capacity for glucose uptake in these transformed cells was enhanced much more than the capacity of galactose uptake. It was concluded that cells infected by the Bryan strain of rous sarcoma virus in the process of transformation selectively synthesize more sites specific for glucose uptake. Lower levels of adenosine triphosphate found in transformed cells possibly contribute to a chronic derepression of uptake sites.

Scaled Quantum Mechanical scale factors for vibrational calculations using alternate polarized and augmented basis sets with the B3LYP density functional calculation model.

The Scaled Quantum Mechanical (SQM) method of scaling calculated force constants to predict theoretically calculated vibrational frequencies is expanded to include a broad array of polarized and augmented basis sets based on the split valence 6-31G and 6-311G basis sets with the B3LYP density functional. Pulay's original choice of a single polarized 6-31G(d) basis coupled with a B3LYP functional remains the most computationally economical choice for scaled frequency calculations. But it can be improved upon with additional polarization functions and added diffuse functions for complex molecular systems. The new scale factors for the B3LYP density functional and the 6-31G, 6-31G(d), 6-31G(d,p), 6-31G+(d,p), 6-31G++(d,p), 6-311G, 6-311G(d), 6-311G(d,p), 6-311G+(d,p), 6-311G++(d,p), 6-311G(2d,p), 6-311G++(2d,p), 6-311G++(df,p) basis sets are shown. The double d polarized models did not perform as well and the source of the decreased accuracy was investigated. An alternate system of generating internal coordinates that uses the out-of plane wagging coordinate whenever it is possible; makes vibrational assignments via potential energy distributions more meaningful. Automated software to produce SQM scaled vibrational calculations from different molecular orbital packages is presented.

Metrics and mappings: a framework for understanding real-world quantitative estimation.

Estimation is influenced by a variety of processes: application of heuristics, domain-specific reasoning, and intuitive statistical induction, among them. In this article, we propose the metrics and mapping framework to account for how these processes are integrated to generate estimates. This framework identifies 2 types of information as critical: knowledge of distributional properties (metric knowledge) and knowledge of relative status of individual entities within the distribution (mapping knowledge). Heuristics and domain-specific knowledge are both viewed as cues that contribute to mapping knowledge; intuitive statistical induction is viewed as providing cues to metric properties. Results of 4 experiments illustrate the framework's usefulness for integrating these types of information and for predicting when people emphasize heuristics and when they emphasize domain-specific knowledge.

Reasoning about geography.

To understand the nature and etiology of biases in geographical judgments, the authors asked people to estimate latitudes (Experiments 1 and 2) and longitudes (Experiments 3 and 4) of cities throughout the Old and New Worlds. They also examined how people's biased geographical judgments change after they receive accurate information ("seeds") about actual locations. Location profiles constructed from the pre- and postseeding location estimates conveyed detailed information about the representations underlying geography knowledge, including the subjective positioning and subregionalization of regions within continents; differential seeding effects revealed between-region dependencies. The findings implicate an important role for conceptual knowledge and plausible-reasoning processes in tasks that use subjective geographical information.

Context memory and the selection of frequency estimation strategies.

When people estimate event frequency, they sometimes retrieve and count event instances. This study demonstrates a direct relation between the use of these enumeration-based strategies and the contents of memory. In 3 experiments, participants studied target-context word pairs, estimated presentation frequency for target words, and recalled context words. Study time, target-context relatedness, and study-phase instructions were manipulated, producing large differences in memory for context words. When context memory was best, estimation time increased sharply with presentation frequency, and the steepness of this estimation time-presentation frequency function decreased with context memory. These results indicate that enumeration was common only when context memory was good, that encoding factors determine how frequency is represented, and that the contents of memory restrict strategy selection.

Updating geographical knowledge: principles of coherence and inertia.

In 2 experiments, the authors investigated how representations of global geography are updated when people learn new location information about individual cities. Participants estimated the latitude of cities in North America (Experiment 1) and in the Old and New Worlds (Experiment 2). After making their first estimates, participants were given information about the latitudes of 2 cities and asked to make a second set of estimates. Both the first and second estimates revealed evidence for psychologically distinct geographical subregions that were coordinated, in an ordinal sense, across the Atlantic Ocean. Further, the second estimates were affected by the nature of the physical adjacency between regions (e.g., the southern U.S. and Mexico) and by accurate location information about distant, but coordinated, subregions (e.g., the southern U.S. and Mediterranean Europe). The data provide support for a framework for making geographical estimates in which people strike a balance between 2 principles: the need to keep their knowledge base coherent, and the inertial tendency to resist changing the knowledge base unless it is necessary to maintain coherence.

Long-term benefits of seeding the knowledge base.

Exposure to numerical examples (seed facts) produced a substantial long-term reduction in domain-specific innumeracy. In particular, learning the populations of 24 seed countries improved accuracy of estimates of the populations of 75 untrained countries, both at the time of learning and 4 months later. Consistent with abstraction-based theories of learning and memory, the benefits of having been exposed to the seed facts were as large 4 months after the exposure as immediately after it, despite the specific populations of the seed countries having been forgotten during the interval.

Estimating the frequency of nonevents: the role of recollection failure in false recognition.

Participants studied lists of multiply presented converging associates (e.g., bed, dream, pillow, etc.) and were timed as they estimated how often they saw list items, related foils (e.g., blanket), and nonpresented critical items (SLEEP). Average number of repetitions (few [3] vs. many [6]) and repetition variability (fixed vs. variable) were manipulated between subjects. Participants responded more slowly to critical items (3.18 sec) than to list items (2.45 sec) or foils (2.22 sec). In addition, critical-item judgments of frequency (JOFs) were about as large as list-item JOFs, and false recognition (i.e., nonzero JOFs) of critical items was most likely in the few-fixed condition (96%) and least likely in the many-fixed condition (74%). These findings suggest that people can use recollection failure--the absence of an anticipated recollective experience, coupled with strong familiarity--to distinguish critical items from list items and that recollection failure is weighted most heavily when people expect familiar probes to access episodic information.

False memories and semantic lexicon arrangement.

A description of semantic lexicon arrangement is a central goal in examinations of language processing. There are a number of ways in which this description has been cast and a host of different mechanisms in place for providing operational descriptions (e.g., feature sharing, category membership, associations, and co-occurrences). We first review two views of the structure of semantic space and then describe an experiment that attempts to adjudicate between these two views. The use of a false memory paradigm provides us with evidence that supports the notion that the semantic lexicon is arranged more by association than by categories or features.

Subjective organization of U.S. presidents.

College students appear to subjectively organize U.S. presidents into three groups and to use this organization to help them learn new information. Results of a paired comparison task in Experiment 1 suggested that subjects organized the presidents into Founding Fathers (Washington through John Quincy Adams), post-World War II presidents (Truman through Reagan), and noncontemporary presidents (Jackson through Franklin Roosevelt). This hypothesized organization was tested in Experiment 2 by presenting subjects with several lists with alternative organizations of the presidents. Difficulty of learning classifications of particular presidents within each list was predicted on the basis of the relation between that president's classification in the list and in the hypothesized preexisting organization. Instances where the preexisting and new classifications were consistent both for the particular president and for all other members of the group were easiest to learn. Instances where the particular president's classification was consistent but where some other presidents in the group were classified differently were of intermediate difficulty. Instances where neither the individual president's classification nor that of the entire group were consistent were most difficult. The results provided converging evidence for the hypothesized subjective organization of the presidents and also demonstrated the usefulness of a method that may be applicable to testing subjective organizations in a wide variety of other areas.

The role of availability in the estimation of national populations.

In two parallel experiments, conducted 17 months apart, we examined the relations among population estimates, availability, and media coverage of the 100 countries with populations of 4 million or more. The results were consistent with the main hypothesis, that availability influences population estimates. Specifically, we found that (1) rated knowledge of each country (our measure of availability) was strongly correlated with estimated population for that country; (2) rated knowledge was more strongly correlated with estimated population than were other plausible predictors, such as land area; (3) a strong partial correlation between rated knowledge and estimated population remained even after true population and true land area were partialed out; and (4) longitudinal changes in rated knowledge predicted longitudinal changes in estimated population. Also as hypothesized, media coverage was positively related to availability and to population estimates. Further, we found that population estimates, rated knowledge, and amount of media coverage that each country received were very stable over the year-and-a-half period. These results led us to consider when availability is most likely to be relied upon heavily in quantitative estimation and how it may be integrated with other knowledge to derive quantitative estimates.

Cytoplasmic vacuoles of Rous virus transformed cells are organelles involved in cation uptake.

Cytoplasmic vacuoles induced during transformation of cells by Bryan strain Rous sarcoma virus (RSV-BH) have been studied using the cationic dye, neutral red(NR). Both the rate of uptake and the accumulation of NR are greater in RSV-BH transformed cells than non-transformed cells however, uptake was greater in vacuolated than in non-vacuolated cells, whether or not they were transformed. The NR was incorporated into pre-existing vacuoles in the absence of cytoplasmic staining, suggesting the existence of direct channels from the cell surface to the vacuoles. Other low mol. wt. cationic dyes could also be incorporated into vacuoles, although those with branched structures or cationic weights greater than 330 were excluded. No anionic dyes were incorporated. Infection of cells with a virus mutant, RSV-BH-Ta, induces temperature-dependent vacuolization. After a shift to the vacuole-permissive temperature, vacuoles developed at different rates and with morphological variations with different cations. Vacuoles which had formed in the presence of several cations, (K+, Rb+, tris+, choline+) failed to disappear when cells were incubated at a temperature sufficient to revert vacuoles formed in Na+-containing medium. No short-term effects of Cl-replacements (Br-, I-, or SO2-4) on vacuolization or reversal were observed. The results suggest that these vacuoles are organelles involved in cation uptake. A possible function for these organelles in RSV-BH induced malignancy is discussed.

Seeds aren't anchors.

Exposure to a few task-relevant numerical facts (seed facts) often improves subsequent numerical estimates. We performed two experiments to investigate the mechanism that produces these seeding effects. In Experiment 1, participants estimated national populations; in Experiment 2, they estimated between-city distances. In both, items were selected so that the actual value of the seed facts (SA) was, on average, below participants' initial estimates for those items (S1) and above the initial estimates for the transfer items (T1). Given this configuration, the anchoring position predicts that the postseeding transfer estimates should be greater than the preseeding transfer estimates (T2 > T1), whereas the feedback/induction position predicts the opposite (T2 < T1). In both experiments, the latter pattern of results emerged, supporting the conclusion that seeds aren't anchors.