RESUMO
The determination of structures for proteins that control the eukaryotic cell cycle by nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography has made a significant contribution to our understanding of the molecular mechanisms that control cell cycle progression. CDK2 has proved particularly tractable to structural analysis, and CDK2 in complex with various regulatory proteins and in different phosphorylation states provides a paradigm for the control of this important kinase family. This chapter describes a number of protocols that can be used to prepare CDKs and selected CDK binding proteins suitable for structural studies by heterologous expression in either E. coli or insect cells.
Assuntos
Proteínas de Ciclo Celular/isolamento & purificação , Ciclo Celular/fisiologia , Animais , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/genética , Quinases relacionadas a CDC2 e CDC28/isolamento & purificação , Bovinos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Cristalografia por Raios X , Ciclina A/química , Ciclina A/genética , Ciclina A/isolamento & purificação , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/isolamento & purificação , Quinases Ciclina-Dependentes/fisiologia , Escherichia coli/genética , Células Eucarióticas , Humanos , Modelos Moleculares , Complexos Multiproteicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Spodoptera , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificaçãoRESUMO
CDK2 and CDK4 known promoter of cell cycling catalyze phosphorylation of RB protein. Enzyme specificity between two CDKs that work at a different cell cycle phase is not clearly understood. In order to define kinase properties of CDK2 and CDK4 in complex with cycline A or cycline D1 in relation to their respective role in cell cycling regulation, we examined enzymatic properties of both CDK4/cycline D1 and CDK2/cycline A in vitro. Association constant, Km for ATP in CDK4/cyclin D1 was found as 418 microM, a value unusually high whereas CDK2/cyclin A was 23 microM, a value close to most of other regulatory protein kinases. Turnover value for both CDK4/cyclin D1 and CDK2/cyclin A were estimated as 3.4 and 3.9 min(-1) respectively. Kinetic efficiency estimation indicates far over one order magnitude less efficiency for CDK4/cyclin D1 than the value of CDK2/cycline A (9.3 pM(-1) min(-1) and 170 pM(-1) min(-1) respectively). In addition, inhibition of cellular CDK4 caused increase of cellular levels of ATP, even though inhibition of CDK2 did not change it noticeably. These data suggest cellular CDK4/cyclin D1 activity is tightly associated with cellular ATP concentration. Also, analysis of phosphorylated serine/threonine sites on RB catalyzed by CDK4/cyclin D1 and CDK2/cyclin A showed significant differences in their preference of phosphorylation sites in RB C-terminal domain. Since RB is known to regulate various cellular proteins by binding and this binding is controlled by its phosphorylation, these data shown here clearly indicate significant difference in their biochemical properties between CDK4/cyclin D1 and CDK2/cyclin A affecting regulation of cellular RB function.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclina A/metabolismo , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas Proto-Oncogênicas , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Baculoviridae/genética , Quinases relacionadas a CDC2 e CDC28/genética , Quinases relacionadas a CDC2 e CDC28/isolamento & purificação , Ciclina A/genética , Ciclina A/isolamento & purificação , Ciclina D1/genética , Ciclina D1/isolamento & purificação , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/isolamento & purificação , Humanos , Cinética , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
ROT1 is an essential gene whose inactivation causes defects in cell cycle progression and morphogenesis in budding yeast. Rot1 affects the actin cytoskeleton during the cell cycle at two levels. First, it is required for the maintenance of apical growth during bud growth. Second, Rot1 is necessary to polarize actin cytoskeleton to the neck region at the end of mitosis; because of this defect, rot1 cells do not properly form a septum to complete cell division. The inability to polarize the actin cytoskeleton at the end of mitosis is not due to a defect in the recruitment of the polarisome scaffold protein Spa2 or the actin cytoskeleton regulators Cdc42 and Cdc24 in the neck region. Previous results indicate a connection between Rot1 and the cyclin Clb2. In fact, overexpression of CLB2 is toxic when ROT1 is partially inactivated, and reciprocally, deletion of CLB2 suppresses the lethality of the rot1 mutant, which indicates a functional antagonism between Clb2 and Rot1. Several genetic interactions suggest a link between Rot1 and the ubiquitin-proteasome system and we show that the Clb2 cyclin is not properly degraded in rot1 cells.
Assuntos
Actinas/metabolismo , Ciclina B/metabolismo , Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Actinas/isolamento & purificação , Quinases relacionadas a CDC2 e CDC28/genética , Quinases relacionadas a CDC2 e CDC28/isolamento & purificação , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclo Celular , Polaridade Celular , Ciclina B/isolamento & purificação , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas de Membrana/genética , Chaperonas Moleculares , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
A partial cDNA fragment of the Cryptococcus neoformans homologue of the main cell cycle control gene CDC28/cdc2 was isolated using degenerate primer RT-PCR. A subsequent search in the C. neoformans genome database identified several sequences similar to CDC28/cdc2. A part of the sequence which showed the highest similarity to CDC28/cdc2 turned out to be identical to the partial cyclin-dependent kinase (Cdk) cDNA fragment isolated by degenerate RT-PCR. The full-length coding region of this Cdk homologue was amplified by RT-PCR using primers designed to target regions around start and stop codons, and the gene was named CnCdk1. To determine its function, an analysis of deduced amino acid sequence of the CnCdk1 was performed and its ability to rescue Saccharomyces cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2- CnCdk1 construct exhibited growth at 36.5 degrees C in galactose-raffinose medium, but not in glucose medium. Results of the sequence analysis and the fact that CnCdk1 is able to complement the S. cerevisiae cdc28-ts mutation support its assumed role as the CDC28/cdc2 homologue in C. neoformans.