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.

Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases.

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. Here we identify indirubin and its analogues as potent inhibitors of cyclin-dependent kinases (CDKs). The crystal structure of CDK2 in complex with indirubin derivatives shows that indirubin interacts with the kinase's ATP-binding site through van der Waals interactions and three hydrogen bonds. Indirubin-3'-monoxime inhibits the proliferation of a large range of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. These results have implications for therapeutic optimization of indigoids.

Age transitions in the course of bipolar I disorder.

BACKGROUND: This analysis aimed to show whether symptoms of either pole change in their persistence as individuals move through two decades, whether such changes differ by age grouping, and whether age of onset plays an independent role in symptom persistence. METHOD: Participants in the National Institute of Mental Health (NIMH) Collaborative Depression Study (CDS) who completed at least 20 years of follow-up and who met study criteria for bipolar I or schizo-affective manic disorder, before intake or during follow-up, were divided by age at intake into youngest (18-29 years, n=56), middle (30-44 years, n=68) and oldest (>44 years, n=24) groups. RESULTS: The persistence of depressive symptoms increased significantly in the two younger groups. Earlier ages of onset were associated with higher depressive morbidity throughout the 20 years of follow-up but did not predict changes in symptom persistence. The proportions of weeks spent in episodes of either pole correlated across follow-up periods in all age groupings, although correlations were stronger for depressive symptoms and for shorter intervals. CONCLUSIONS: Regardless of age at onset, the passage of decades in bipolar illness seems to bring an increase in the predominance of depressive symptoms in individuals in their third, fourth and fifth decades and an earlier age of onset portends a persistently greater depressive symptom burden. The degree to which either depression or manic/hypomanic symptoms persist has significant stability over lengthy periods and seems to reflect traits that manifest early in an individual's illness.

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.

The association between course of illness and subsequent morbidity in bipolar I disorder.

OBJECTIVE: We examined the relationship between certain bipolar I disorder clinical course variables over 5 years with outcome over the subsequent 5-year period. METHODS: Prospective observational follow-up data of 123 bipolar I subjects were analyzed. Predictive clinical variables included the frequency and direction of switches, and the quantity, polarity and length of affective periods. Outcome variables were an affective burden index (ABI) accounting for week-by-week severity and weeks hospitalized. Bivariate analyses guided the selection of predictors for multivariable analyses against the outcome variables. RESULTS: Affective burden index: while the number and direction of switches, the number of polyphasic episodes, weeks in hypomania/mania/mixed state, weeks in minor/major depression, weeks in at least marked affective syndrome, and weeks in any affective syndrome all had bivariate correlation (p<0.01) with the ABI, only weeks in hypomania/mania/mixed state and weeks in minor/major depression made significant contributions in the multivariable analysis (p<0.01) with the ABI. Weeks hospitalized: weeks in at least marked affective syndrome were significantly correlated with weeks hospitalized in bivariate analysis (p<0.01), and maintained a contribution to weeks hospitalized in the multivariable analysis (p<0.01). CONCLUSIONS: The quantity and severity of weeks in symptomatic affective states are possibly greater predictors of affective burden in bipolar I patients than the quantity and direction of affective switches.

Simultaneous expression of two P-glycoprotein genes in drug-sensitive Chinese hamster ovary cells.

Overexpression of P-glycoprotein is characteristic of multidrug-resistant cells. We analyzed four P-glycoprotein transcripts that are simultaneously expressed in a drug-sensitive Chinese hamster ovary cell line. We concluded that these transcripts are encoded by two distinct members of a P-glycoprotein multigene family, each of which has two alternative polyadenylation sites. A comparison of the two hamster sequences with the single reported human and mouse P-glycoprotein cDNA sequences demonstrates that P-glycoprotein is a highly conserved protein, that the hamster multigene family is undergoing concerted evolution, and that differences between gene family members are maintained across species. These conserved differences suggest that there may be functional differences between P-glycoprotein molecules.

Cyclin-dependent kinases: inhibition and substrate recognition.

Four unresolved issues of cyclin-dependent kinase (CDK) regulation have been addressed by structural studies this year - the mechanism of CDK inhibition by members of the INK4 family of CDK inhibitors, consensus substrate sequence recognition by CDKs, the role of the cyclin subunit in substrate recognition and the structural mechanism underlying CDK inhibition by phosphorylation.

The cell cycle and suc1: from structure to function?

Structures have recently been determined for the yeast Schizosaccharomyces pombe cell cycle regulatory protein, CKS/suc1, and its human equivalent. The structures provide some long-awaited clues about the role of CKS/suc1 in cell cycle control.

Structural principles in cell-cycle control: beyond the CDKs.

Retinoblastoma protein (Rb) interacts with cyclin-dependent kinases and regulates the transcription of genes necessary for progression through the S phase of the cell cycle. Clues to the atomic mechanisms involved are offered by the structure of the two pocket regions of Rb in complex with a short peptide from a viral oncoprotein. Structures of cyclins, Rb and TFIIB reveal that a common motif occurs in proteins regulating three consecutive events of cell-cycle control.

Inhibitor binding to active and inactive CDK2: the crystal structure of CDK2-cyclin A/indirubin-5-sulphonate.

BACKGROUND: Cyclin-dependent kinase 2 (CDK2) is an important target for structure-based design of antitumor agents. Monomeric CDK2 is inactive. Activation requires rearrangements to key structural elements of the enzyme's active site, which accompany cyclin binding and phosphorylation. To assess the validity of using monomeric CDK2 as a model for the active kinase in structure-based drug design, we have solved the structure of the inhibitor indirubin-5-sulphonate (E226) complexed with phospho-CDK2-cyclin A and compared it with the structure of E226 bound to inactive, monomeric CDK2. RESULTS: Activation of monomeric CDK2 leads to a rotation of its N-terminal domain relative to the C-terminal lobe. The accompanying change in position of E226 follows that of the N-terminal domain, and its interactions with residues forming part of the adenine binding pocket are conserved. The environment of the ATP-ribose site, not explored by E226, is significantly different in the binary complex compared to the monomeric complex due to movement of the glycine loop. Conformational changes also result in subtle differences in hydrogen bonding and electrostatic interactions between E226's sulphonate and CDK2's phosphate binding site. Affinities calculated by LUDI for the interaction of E226 with active or inactive CDK2 differ by a factor of approximately ten. CONCLUSIONS: The accuracy of monomeric CDK2 as an inhibitor design template is restricted to the adenine binding site. The general flexibility observed for the glycine loop and subtle changes to the phosphate binding site suggest a need to study interactions between inhibitors and active CDK2 in structure-based drug design programs.

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.

Chemical inhibitors of cyclin-dependent kinases: insights into design from X-ray crystallographic studies.

Cyclin-dependent kinases (CDKs) are a family of protein kinases that regulate progression through the eukaryotic cell cycle. Aberrant CDK activity or function is a common defect in human tumours, resulting in unrestrained cellular proliferation. X-ray crystallographic analysis of monomeric CDK2 and CDK2 complexes has revealed how phosphorylation and cyclin binding mediate enzyme activation and how this activity can be regulated by further protein association. Current research aims to improve the selectivity and/or potency of small molecule CDK inhibitors, both to develop specific probes to study the roles of the different CDK family members in coordinating cell cycle progression, and as lead molecules for the design of therapeutically useful drugs. This design process has been assisted by the availability of a number of CDK2/inhibitor structures determined using X-ray crystallography. These structures have shown that molecules related to ATP can be accommodated in the ATP-binding site in a number of orientations, utilising interactions observed between CDK2 and its natural ligand, as well as novel interactions with CDK2 residues that lie both within and outside the active site cleft. This site can also bind inhibitors that are structurally unrelated to ATP. These results suggest that it may be possible to design pharmacologically and pharmaceutically important ATP-binding site-directed ligands that act as specific and potent inhibitors of CDK activity.

The psychotic spectrum: validity and reliability of the Structured Clinical Interview for the Psychotic Spectrum.

This study evaluates the validity and the reliability of a new instrument developed to assess the psychotic spectrum: the Structured Clinical Interview for the Psychotic Spectrum (SCI-PSY). The instrument is based on a spectrum model that emphasizes soft signs, low-grade symptoms, subthreshold syndromes, as well as temperamental and personality traits comprising the clinical and subsyndromal psychotic manifestations. The items of the interview include, in addition to a subset of the DSM-IV criteria for psychotic syndromes, a number of features derived from clinical experience and from a review of the phenomenological descriptions of psychoses. Study participants were enrolled at 11 Italian Departments of Psychiatry located at 9 sites and included 77 consecutive patients with schizophrenia or schizoaffective disorder, 66 with borderline personality disorder, 59 with psychotic mood disorders, 98 with non-psychotic mood disorders and 57 with panic disorder. A comparison group of 102 unselected controls was enrolled at the same sites. The SCI-PSY significantly discriminated subjects with any psychiatric diagnosis from controls and subjects with from those without psychotic disorders. The hypothesized structure of the instrument was confirmed empirically.

Major depression in a nonclinical sample. Demographic and clinical risk factors for first onset.

The relatives, controls, and spouses of affectively ill probands underwent diagnostic examinations on two occasions, 6 years apart. Of 965 subjects who had never been mentally ill when first examined, 11.8% had development of at least one episode of major depression as defined by the Research Diagnostic Criteria during the ensuing 6 years. Subjects younger than 40 years were three times more likely than older subjects to develop depression and women were approximately twice as likely as men to develop depression regardless of age. Marital disruption, a farm setting, and high educational achievement substantially increased the risk of depression among female subjects. Of 214 never-depressed subjects with a history of nonaffective mental disorder, 62 (29.0%) developed major depression. Age and sex were again powerful determinants. The course of prospectively observed secondary depression was more severe than that for primary depression.

Rapidly cycling affective disorder. Demographics, diagnosis, family history, and course.

Of 919 patients with major affective disorders who completed at least 1 year of a 5-year, semiannual follow-up, 45 developed a rapidly cycling bipolar course during the first year, but only one developed a rapidly cycling unipolar course. In comparison with patients who showed a non-rapidly cycling bipolar course, those who became rapid cyclers were more likely to be female and to have exhibited depression, hypomania, or cycling between depression and hypomania within the index episode. Family study data revealed no evidence that high cycle frequencies breed true. Rapid cycling was associated with a significantly lower likelihood of recovery in the second year of follow-up but not in the third, fourth, or fifth. These data suggest that rapid cycling is, in the large majority of cases, a transient, nonfamilial manifestation of bipolar affective disorder.

Premenstrual depressive changes. Value of differentiation.

Premenstrual depressive changes and differential correlates of specific subtypes of premenstrual dysphoria vary. Our data support two basic assumptions: (1) Premenstrual changes should be studied as diversified subtypes rather than as a single premenstrual tension syndrome; such an approach might lead to a better understanding of the pathophysiology of specific types of premenstrual changes. (2) Some specific subtypes of premenstrual changes of a depressive nature resemble some subtypes of affective disorder and, hence, may serve as a model for the study of these disorders.

A diagnostic interview: the schedule for affective disorders and schizophrenia.

The Schedule for Affective Disorders and Schizophrenia (SADS) was developed to reduce information variance in both the descriptive and diagnostic evaluation of a subject. The SADS is unique among rating scales in that it provides for (1) a detailed description of the features of the current episodes of illness when they were at their most severe; (2) a description of the level of severity of manifestations of major dimensions of psychopathology during the week preceding the evaluation, which can then be used as a measure of change; (3) a progression of questions and criteria, which provides information for making diagnoses; and (4) a detailed description of past psychopathology and functioning relevant to an evaluation of diagnosis, prognosis, and overall severity of disturbance. This article reports on initial scale development and reliability studies of the items and the scale scores.