RESUMO
Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases (Cdk/cyclins), thus triggering initiation and progression of successive phases of the cell cycle. In our efforts to elucidate the interaction between Cdc25B and the natural substrate, bis-phosphorylated Cdk2/CycA (Cdk2-pTpY/CycA), we have previously found that the 17 residues of the C-terminal tail mediate a factor of 10 in substrate recognition. In the studies reported here, we localize the majority of this interaction using site-directed mutagenesis to two arginine residues (Arg556 and Arg562) located within this C-terminal region. We also show that the catalytic domain of Cdc25C, which differs most significantly from Cdc25B in this tail region, has a 100-fold lower activity toward Cdk2-pTpY/CycA. We further demonstrate that the proper presentation of the C-terminal tail of Cdc25B can be achieved in a "gain-of-function" chimeric protein consisting of the C-terminal tail of Cdc25B fused onto the catalytic core of Cdc25C. The >10-fold increase in activity seen only in the chimeric protein containing the two critical arginine residues demonstrates that the modular C-terminal tail of Cdc25B is the basis for most of the catalytic advantage of Cdc25B versus Cdc25C toward the Cdk2-pTpY/CycA substrate.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular/metabolismo , Fosfatases cdc25/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/agonistas , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/metabolismo , Fosfopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Fosfatases cdc25/agonistas , Fosfatases cdc25/antagonistas & inibidores , Fosfatases cdc25/genéticaRESUMO
Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases, thus causing initiation and progression of successive phases of the cell cycle. Although it is not significantly structurally homologous to other well-characterized members, Cdc25 belongs to the class of well-studied cysteine phosphatases as it contains their active site signature motif. However, the catalytic acid needed for protonation of the leaving group has yet to be identified. To elucidate the role and identity of this key catalytic residue, we have performed a detailed pH-dependent kinetic analysis of Cdc25B. The pK(a) of the catalytic cysteine was found to be 5.6-6.3 in steady state and one-turnover burst experiments using the small molecule substrates p-nitrophenyl phosphate and 3-O-methylfluorescein phosphate. Interestingly, Cdc25B does not exhibit the typical bell-shaped pH-rate profile with small molecule substrates seen in other cysteine phosphatases and indicative of the catalytic acid because it lacks pH dependence between 6.5 and 9. Reactions of Cdc25B with the natural substrate Cdk2-pTpY/CycA, however, did yield a bell-shaped pH-rate profile with a pK(a) of 6.1 for the catalytic acid residue. Recent structural studies of Cdc25 have suggested that Glu474 [Fauman, E. B., et al. (1998) Cell 93, 617-625] or Glu478 [Reynolds, R. A., et al. (1999) J. Mol. Biol. 293, 559-568] could function as the catalytic acid in Cdc25B. Using site-directed mutagenesis and truncation experiments, however, we found that neither of these residues, nor the unstructured C-terminus, is responsible for the observed pH dependence. These results indicate that the catalytic acid does not appear to lie within the known structure of Cdc25B and may lie on its protein substrate.
Assuntos
Domínio Catalítico , Proteínas de Ciclo Celular/metabolismo , Proteínas de Xenopus , Fosfatases cdc25/metabolismo , Substituição de Aminoácidos/genética , Animais , Catálise , Domínio Catalítico/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Vetores Genéticos/síntese química , Ácido Glutâmico/genética , Glutamina/genética , Concentração de Íons de Hidrogênio , Cinética , Mutagênese Sítio-Dirigida , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Especificidade por Substrato , Xenopus laevis , Fosfatases cdc25/biossíntese , Fosfatases cdc25/genética , Fosfatases cdc25/isolamento & purificaçãoRESUMO
Newborn screening for the hemoglobinopathies has been shown to reduce morbidity and mortality, particularly for sickle cell anemia, by facilitating initiation of penicillin prophylaxis by 4 months of age. The purpose of the current investigation was to determine whether molecular genetic follow-up testing could be introduced into a neonatal hemoglobinopathy screening program and, if successfully introduced, whether it would reduce time to diagnostic confirmation. Between July 1, 1991, and October 7, 1992, 518 original dried blood specimens were referred from the Texas Department of Health Neonatal Hemoglobinopathy Screening Program for molecular genetic follow-up testing. Allele-specific cleavage (ASC) after amplification with matched and mismatched polymerase chain reaction primers was compared to allele-specific oligonucleotide (ASO) hybridization. By November 2, 1992, molecular genetic analyses were definitive in 506, and agreement was observed between ASC and ASO hybridization in all specimens analyzed. Approximately 13% of those initially screened FS were considered probable S/beta-thal by DNA and RNA testing. Rapid molecular genetic analysis contributed to a substantial reduction of the mean age at confirmation by approximately 50%, to about 2 months of age. ASC is a reliable method for molecular genetic analysis of dried blood specimens, providing methodology which can be readily automated. An automated method is demonstrated that is based on microtiter plate technology and will significantly reduce labor intensity and costs, while increasing sample throughput. Even with current manual testing methods, DNA and RNA analysis of initial newborn screening specimens will reduce the age at confirmation well under 4 months, the age cut-off for effective initiation of penicillin prophylaxis.