The activation of most protein kinases requires phosphorylation at a conserved site within a structurally defined segment termed the activation loop. A classic example is the regulation of the cell cycle control enzyme, CDK2/cyclin A, in which catalytic activation depends on phosphorylation at Thr 160 in CDK2. The structural consequences of phosphorylation have been revealed by x-ray crystallographic studies on CDK2/cyclin A and include changes in conformation, mainly of the activation loop. Here, we describe the kinetic basis for activation by phosphorylation in CDK2/cyclin A. Phosphorylation results in a 100,000-fold increase in catalytic efficiency and an approximate 1,000-fold increase in the overall turnover rate. The effects of phosphorylation on the individual steps in the catalytic reaction pathway were determined using solvent viscosometric techniques. It was found that the increase in catalytic power arises mainly from a 3,000-fold increase in the rate of the phosphoryl group transfer step with a more moderate increase in substrate binding affinity. In contrast, the rate of phosphoryl group transfer in the ATPase pathway was unaffected by phosphorylation, demonstrating that phosphorylation at Thr 160 does not serve to stabilize ATP in the ATPase reaction. Thus, we hypothesize that the role of phosphorylation in the kinase reaction may be to specifically stabilize the peptide phosphoacceptor group. Cellular proliferation is controlled by a family of protein kinases in which the catalytic subunits are members of the cyclin-dependent kinase (CDK) 1 family and the regulatory subunits are cyclins. To date, nine distinct CDKs in addition to eight different cyclins have been identified, in which different CDK/cyclin combinations serve to regulate distinct points in the mammalian cell division cycle. Although Cdc2 (CDK1)/ cyclin B controls the transition of cells from the G 2 to M-phase, the activities of CDK2/cyclin E and CDK2/cyclin A are critical for G 1 /S-phase transition and progression through S-phase, respectively (1). Since the critical role of the CDKs in cell cycle control has been well established, understanding the details of their regulation is now of fundamental importance.The three-dimensional structures of several forms of CDK2 have been solved by x-ray crystallography. Like all protein kinases, CDK2 displays a globular fold consisting of two lobes, a smaller N-terminal lobe that is principally ␤-sheet and a larger C-terminal lobe that is principally ␣-helix. The bilobal interface constitutes the active site cleft into which the adenine ring of substrate ATP is deeply buried. The ATP ␥-phosphate is directed toward the mouth of the active site where peptide and protein substrates bind and where phosphoryl group transfer occurs (for a review see Ref.2). Located near the mouth of the active site is a conserved loop structure termed the activation loop (residues 146 -166). This loop structure is present in all protein kinases (3), and phosphorylation at a conserved site within the activation loop ...

Activation of the cyclin-dependent kinases is a two-step process involving cyclin binding followed by phosphorylation at a conserved threonine residue within the kinase activation loop. In this study, we describe the separate roles of cyclin A binding versus phosphorylation in the overall activation mechanism of CDK2. Interaction of CDK2 with cyclin A results in a partially active complex that is moderately defective in the binding of the protein substrate, but not ATP, and severely defective in both phosphoryl group transfer and turnover. Alternatively, phosphorylation of the CDK2 monomer also results in a partially activated species, but one that is severely (> or = 480-fold) defective in substrate binding exclusively. Catalytic turnover in the phosphorylated CDK2 monomer is largely unimpaired (approximately 8-fold lower). Our data support a model for the activation of CDK2 in vivo, in which interaction of unphosphorylated CDK2 with cyclin A serves to configure the active site for ground-state binding of both ATP and the protein substrate, and further aligns ATP in the transition state for phosphoryl transfer. Optimizing the alignment of protein substrates in the phosphoryl transfer reaction is the principal role of phosphorylation at Thr(160).

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We examined the hypothesis that skilled performance is monitored on the basis of fluency, where fluency is operationally defined as temporal regularity or rhythmicity rather than speed. Since error is often associated with variable timing, we tested the possibility that people use varied timing as a metacognitive cue. Using a sequential counting task, which may be representative of the broader class of skilled, multi-step tasks, we found that shifting between irregular and regular timing led to greater confidence ratings when the timing associated with the task was regular. We argue that regular, consistent timing, when compared directly to irregular timing, produced feelings of fluent task performance, leading to increased confidence. In the first experiment, we demonstrated that both accuracy and confidence were higher when participants completed a task presented with regular timing. In the second experiment, we found a dissociation between accuracy and confidence, strengthening the argument that individuals relied on monitoring of fluency to support their metacognitive judgments. In Study 3 and an assessment of naïve beliefs, we ruled out alternative explanations for these findings.

In some cognitive tasks, coordinating acquisition of information from the environment with ongoing mental processes is relatively straightforward. In these cases, the needed information is continually available in the environment, and the timing of skilled information acquisition activities can be triggered by ongoing mental processes. Reading is one example. The information needed is continually available, and it is typically organized in a well-learned, conventional manner. The mental processes tracking the reading can, therefore, trigger the perceptual and attentional processes needed to pick up the next relevant word, and skilled readers accomplish this coordination with little effort or deliberation (e.g., Just & Carpenter, 1980). However, there are many tasks in which people must take some deliberate action to make necessary information available. While working at a computer, for example, one might have to click a mouse button to display some piece of information. In other cases, visual search might be necessary to find the relevant information. In the present research, we examined how people are able to strategically coordinate their information acquisition activities with ongoing mental processes in the face of cognitive and environmental constraints.The different orders in which individuals pick up information to perform a task are likely to vary in their efficiency. For example, Bovair and Kieras (1991) reviewed evidence that individuals follow procedural instructions more effectively when elements are presented in the order in which they must be used to perform the procedure. They noted that although some evidence supports this principle at the level of elements required for single procedural steps, interpreting previous research on this issue is difficult, because the optimal order of elements is not always obvious and because there may be conflicts between procedural and grammatical orderings for instructions. In the present experiments, we addressed this issue by making use of a paradigm in which there is previous evidence concerning the optimal ordering of procedural elements and in which concerns about textual and grammatical constraints are minimal.Our paradigm is based on previous research that suggests that there is an optimal order in which information should be picked up in arithmetic tasks, such as adding a pair of digits. In this task, one could consider the operator (addition) first, consider the operands (digits) first, or consider these elements in the order operand -operatoroperand. Sohn and Carlson (1998) presented the participants with single-step Boolean and arithmetic tasks in which the elements appeared in each of these orders. For both problem types, the participants were fastest (measured from the point at which all information was available) when they saw the operator first. Carlson and Sohn (2000) demonstrated a similar effect in multiple-step problems in both arithmetic and spatial domains. In these problems, the result of one step served as an operand for the subsequent step...