RNA-driven cyclin-dependent kinase regulation: when CDK9/cyclin T subunits of P-TEFb meet their ribonucleoprotein partners.

The positive transcription elongation factor (P-TEFb) consists of CDK9, a cyclin-dependent kinase and its cyclin T partner. It is required for transcription of most class II genes. Its activity is regulated by non-coding RNAs. The 7SK cellular RNA turns the HEXIM cellular protein into a P-TEFb inhibitor that binds its cyclin T subunit. Thus, P-TEFb activity responds to variations in global cellular transcriptional activity and to physiological conditions linked to cell differentiation, proliferation or cardiac hypertrophy. In contrast, the Tat activation region RNA plays an activating role. This feature at the 5' end of the human immunodeficiency (HIV) viral transcript associates with the viral protein Tat that in turn binds cyclin T1 and recruits active P-TEFb to the HIV promoter. This results in enhanced P-TEFb activity, which is critical for an efficient production of viral transcripts. Although discovered recently, the regulation of P-TEFb becomes a paradigm for non-coding RNAs that regulate transcription factors. It is also a unique example of RNA-driven regulation of a cyclindependent kinase.

Structural Basis for Interaction of Inhibitors with Cyclin-Dependent Kinase 2

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular model of cyclin-dependent kinase 5 complexed with roscovitine

Here is described a structural model for the binary complex CDK5-roscovitine. Roscovitine has been shown to potently inhibit cyclin-dependent kinases 1, 2 and 5 (CDK1, 2, and 5), and the structure of CDK2 complexed with roscovitine has been reported; however, no structural data, are available for complexes of CDK5 with inhibitors. The structural model indicates that roscovitine strongly binds to the ATP-binding pocket of CDK5 and structural comparison of the CDK2-roscovitine complex correlates the structural differences with differences in inhibition of these CDKs by this inhibitor. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known lead structures of adenine derivatives. (C) 2002 Elsevier B.V. (USA). All rights reserved.

Structural basis for inhibition of cyclin-dependent kinase 9 by flavopiridol

Flavopiridol has been shown to potently inhibit CDK1 and 2 (cyclin-dependent kinases 1 and 2) and most recently it has been found that it also inhibits CDK9. The complex CDK9-cyclin T1 controls the elongation phase of transcription by RNA polymerase II. The present work describes a molecular model for the binary complex CDK9-flavopiridol. This structural model indicates that the inhibitor strongly binds to the ATP-binding pocket of CDK9 and the structural comparison of the complex CDK2-flavopiridol correlates the structural differences with differences in inhibition of these CDKs by flavopiridol. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known leading structures such as flavones and adenine derivatives. © 2002 Elsevier Science (USA). All rights reserved.

Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis

Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.

Dynamic interplay between the intermediate filament protein nestin and Cyclin-dependent kinase 5

Cellen har ett s.k. cytoskelett som bl.a. ger stadga åt cellen och deltar i dess form- och rörelsefunktioner. Intermediärfilamenten är en viktig del av cytoskelettet och de har länge varit kända för sina väsentliga roller i att upprätthålla den cellulära organisationen och vävnadernas integritet. På senare år har man insett att intermediärfilamenten har en större funktionell mångsidighet än man tidigare tänkts sig, i och med att en rad olika studier har visat betydelsen av intermediärfilamenten vid olika signaleringprocesser. Dessa proteinnätverk samverkar nämligen med kinaser och andra viktiga signalfaktorer och deltar därmed i cellens signaleringmaskineri. Intermediärfilamentproteinet nestin används ofta som en markör för stamceller men dess fysiologiska funktioner är i stort sett okända. Interaktion mellan nestin och ett signalkomplex bestående av cyklin-beroende kinas 5 (eng. Cyclin-dependent kinase, Cdk5) och dess aktivatorprotein p35 upptäcktes i vårt laboratorium före denna avhandling påbörjades. Därför var syftet med min avhandling att undersöka den funktionella betydelsen av nestin i regleringen av Cdk5/p35 komplexet. Cdk5 är ett multifunktionellt kinas som reglerar både utvecklingen och stressreaktioner i nerver och muskler. Vi visade att nestin skyddar neuronala stamceller under oxidativ stress genom dess förmåga att hämma Cdk5s skadliga aktivitet. Genom att förankra Cdk5/p35 komplexet, reglerar nestin den subcellulära lokaliseringen av Cdk5/p35 och minskar klyvningen av p35 till den mer stabila aktivatorn p25. Vi demonstrerade också aktiveringsmekanismen för Cdk5 under differentiering av muskelceller. Proteinkinas C zeta (PKCzeta) avslöjades ha en förmåga att accelera klyvningen av p35 till p25, och därmed öka aktiviteten hos Cdk5. Nestin kunde genom sin förmåga att reglera Cdk5 signalkomplexet styra muskelcellernas differentiering. Denna doktorsavhandling har på ett avgörande vis ökat förståelsen av de reglerande mekanismer som styr Cdk5 aktivering. Avhandling presenterar nestin och PKCzeta som kritiska faktorer i denna reglering. Vidare innehåller avhandlingen ny information om de cellulära funktionerna hos nestin som vi har visat vara en viktig reglerare av cellernas överlevnad och differentiering.

Cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors: detection methods and activity measurements

The cyclin/cyclin-dependent kinase (Cdk) complexes and the Cdk inhibitors (CDKI) are crucial regulators of cell cycle progression in all eukaryotic cells. Using rat cardiac myocytes as a model system, this chapter provides a detailed account of methods that can be employed to measure both cyclin/Cdk activity in cells and the extent of CDKI inhibitory activity present in a particular cell type.

Cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors: detection methods and activity measurements

The cyclin/cyclin-dependent kinase (Cdk) complexes and the Cdk inhibitors (CDKI) are crucial regulators of cell cycle progression in all eukaryotic cells. Using rat cardiac myocytes as a model system, this chapter provides a detailed account of methods that can be employed to measure both cyclin/Cdk activity in cells and the extent of CDKI inhibitory activity present in a particular cell type.

Downregulation of cyclin-dependent kinase inhibitors p21 and p27 in pressure-overload hypertrophy

We postulated that the cyclin-dependent kinase inhibitors p21 and p27 could regulate the alterations in growth potential of cardiomyocytes during left ventricular hypertrophy (LVH). LVH was induced in adult rat hearts by aortic constriction (AC) and was monitored at days 0, 1, 3, 7, 14, 21, and 42 postoperation. Relative to sham-operated controls (SH), left ventricle (LV) weight-to-body weight ratio in AC increased progressively with time without significant differences in body weight or right ventricle weight-to-body weight ratio. Atrial natriuretic factor mRNA increased significantly in AC to 287% at day 42 compared with SH (P < 0.05), whereas p21 and p27 mRNA expression in AC rats decreased significantly by 58% (P < 0.03) and 40% (P < 0.05) at day 7, respectively. p21 and p27 protein expression decreased significantly from days 3 to 21 in AC versus SH, concomitant with LV adaptive growth. Immunocytochemistry showed p21 and p27 expression in cardiomyocyte nuclei. Thus downregulation of p21 and p27 may modulate the adaptive growth of cardiomyocytes during pressure overload-induced LVH.

Arresting developments in the cardiac myocyte cell cycle: Role of cyclin-dependent kinase inhibitors

Like most other cells in the body, foetal and neonatal cardiac myocytes are able to divide and proliferate. However, the ability of these cells to undergo cell division decreases progressively during development such that adult myocytes are unable to divide. A major problem arising from this inability of adult cardiac myocytes to proliferate is that the mature heart is unable to regenerate new myocardial tissue following severe injury, e.g. infarction, which can lead to compromised cardiac pump function and even death. Studies in proliferating cells have identified a group of genes and proteins that controls cell division. These proteins include cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKIs), which interact with each other to form complexes that are essential for controlling normal cell cycle progression. A variety of other proteins, e.g. the retinoblastoma protein (pRb) and members of the E2F family of transcription factors, also can interact with, and modulate the activities of, these complexes. Despite the major role that these proteins play in other cell types, little was known until recently about their existence and activities in immature (proliferating) or mature (non-proliferating) cardiac myocytes. The reason(s) why cardiac myocytes lose their ability to divide during development remains unknown, but if strategies were developed to understand the mechanisms underlying cardiac myocyte growth, it could open up new avenues for the treatment of cardiovascular disease. In this article, we shall review the function of the cell cycle machinery and outline some of our recent findings pertaining to the involvement of the cell cycle in modulating cardiac myocyte growth and hypertrophy.

Molecular models of cyclin-dependent kinase 1 complexed with inhibitors

Roscovitine and flavopiridol have been shown to potently inhibit cyclin-dependent kinase 1 and 2 (CDK1 and 2). The structures of CDK2 complexed with roscovitine and deschoroflavopiridol have been reported, however no crystallographic structure is available for complexes of CDK1 with inhibitors. The present work describes two molecular models for the binary complexes CDK1:roscovitine and CDK1:flavopiridol. These structural models indicate that both inhibitors strongly bind to the ATP-binding pocket of CDKI and structural comparison of the CDK complexes correlates the structures with differences in inhibition of these CDKs by flavopiridol and roscovitine. This article explains the structural basis for the observed differences in activity of these inhibitors. (C) 2004 Elsevier B.V. All rights reserved.

Impairment of rat postnatal lung alveolar development by glucocorticoids: involvement of the p21CIP1 and p27KIP1 cyclin-dependent kinase inhibitors

It has been shown that glucocorticoids accelerate lung development by limiting alveolar formation resulting from a premature maturation of the alveolar septa. Based on these data, the aim of the present work was to analyze the influence of dexamethasone on cell cycle control mechanisms during postnatal lung development. Cell proliferation is regulated by a network of signaling pathways that converge to the key regulator of cell cycle machinery: the cyclin-dependent kinase (CDK) system. The activity of the various cyclin/CDK complexes can be modulated by the levels of the cyclins and their CDKs, and by expression of specific CDK inhibitors (CKIs). In the present study, newborn rats were given a 4-d treatment with dexamethasone (0.1-0.01 microg/g body weight dexamethasone sodium phosphate daily on d 1-4), or saline. Morphologically, the treatment caused a significant thinning of the septa and an acceleration of lung maturation on d 4. Study of cyclin/CDK system at d 1-36 documented a transient down-regulation of cyclin/CDK complex activities at d 4 in the dexamethasone-treated animals. Analysis of the mechanisms involved suggested a role for the CKIs p21CIP1 and p27KIP1. Indeed, we observed an increase in p21CIP1 and p27KIP1 protein levels on d 4 in the dexamethasone-treated animals. By contrast, no variations in either cyclin and CDK expression, or cyclin/CDK complex formation could be documented. We conclude that glucocorticoids may accelerate lung maturation by influencing cell cycle control mechanisms, mainly through impairment of G1 cyclin/CDK complex activation.

Role of the cyclin dependent kinase-4 in normal and neoplastic proliferation

In the last few years, our laboratory has studied the regulatory mechanisms of proliferation and differentiation in epidermal tissues. Our results showed differences in the roles of cyclin dependent-kinases 4 and 6, and the three D-type cyclins, during normal epidermal proliferation and neoplastic development. Thus, to elucidate the role of the different cell cycle regulators, we developed transgenic mice that overexpress CDK4 (K5-CDK4), or their cognate D-type cyclins, in epithelial tissues. The most severe phenotype was observed in K5-CDK4 animals that developed dermal fibrosis, epidermal hyperplasia and hypertrophy. Forced expression of CDK4 in the epidermal basal cell layer increased the malignant conversion of skin papillomas to squamous cell carcinomas (SCC). Contrastingly, lack of CDK4 completely inhibited tumor development, suggesting that CDK4 is required in this process. Biochemical studies demonstrated that p21 Cip1 and p27Kip1 inhibitors are sequestered by CDK4 resulting in indirect activation of Cyclin E/CDK2, implicating the non-catalytic activity of CDK4 in deregulation of the cell cycle progression. ^ It has been proposed that the proliferative and oncogenic role of Myc is linked to its ability to induce the transcription of CDK4, cyclin D1, and cyclin D2 in vitro. Deregulation of Myc oncogene has been found in several human cancers. Also it has been demonstrated that CDK4 has the ability to functionally inactivate the product of the tumor suppressor gene Rb, providing a link between Myc and the CDK4/cyclin D1/pRb/p16 pathway in some malignant tumors. Here, we sought to determine the role of CDK4 as a mediator of Myc activities by developing a Myc overexpressing mouse nullizygous for CDK4. We demonstrated that lack of CDK4 results in reduced keratinocyte proliferation and epidermal thickness in K5-Myc/CDK4-null mice. In addition, complete reversion of tumor development was observed. All together, this work demonstrates that CDK4 acts as an oncogene independent of the D-type cyclin levels and it is an important mediator of the tumorigenesis induced by Myc. In addition, we showed that the sequestering activity of CDK4 is critical for the development of epidermal hyperplasia during normal proliferation, malignant progression from papillomas to squamous cell carcinomas, and tumorigenesis induced by Myc. ^

Oncogenic stimulation recruits cyclin-dependent kinase in the cell cycle start in rat fibroblast

The rat fibroblast NRK cells are transformed reversibly by a combination of growth factors. When stimulated with serum, NRK cells rely on cyclin-dependent kinase 4 (Cdk4) for their S phase entry. However, when stimulated with serum containing oncogenic growth factors, they come to rely on either Cdk4 or Cdk6, and their S phase entry cannot be blocked unless both Cdk4 and Cdk6 are immunodepleted. Such change of dependence does not occur in the NRK cell mutants defective in an oncogenic signal pathway and, therefore, deficient in anchorage-independent cell cycle start ability, correlating Cdk6 dependence with this remarkable, cancer-associated phenotype. However, both Cdk4 and Cdk6 are activated upon serum stimulation, and neither the amounts of Cdk6, Cdk4, cyclin D1, and cyclin-dependent kinase inhibitors nor the activities or subcellular localization of Cdk6 and Cdk4 are significantly influenced by oncogenic stimulation. Thus, oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start in a rat fibroblast, but by a mechanism seemingly unrelated to the regulation of the kinase. Given that many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, our results raise the possibility that the oncogenic stimulation-induced anchorage-independent cell cycle start of NRK is elicited by a mechanism similar to the one used for hematopoietic cell proliferation.