RESUMEN
Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Quinasas/metabolismo , Especificidad de Anticuerpos , Quinasa 4 Dependiente de la Ciclina/metabolismo , Activación Enzimática , Células HCT116 , Humanos , Modelos Biológicos , Mutación/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Proteína Fosfatasa 1/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Saccharomyces cerevisiae , Especificidad por SustratoRESUMEN
OBJECTIVE Microcystic meningioma (MM) is a meningioma variant with a multicystic appearance that may mimic intrinsic primary brain tumors and other nonmeningiomatous tumor types. Dynamic susceptibility contrast (DSC) and dynamic contrast-enhanced (DCE) MRI techniques provide imaging parameters that can differentiate these tumors according to hemodynamic and permeability characteristics with the potential to aid in preoperative identification of tumor type. METHODS The medical data of 18 patients with a histopathological diagnosis of MM were identified through a retrospective review of procedures performed between 2008 and 2012; DSC imaging data were available for 12 patients and DCE imaging data for 6. A subcohort of 12 patients with Grade I meningiomas (i.e., of meningoepithelial subtype) and 54 patients with Grade IV primary gliomas (i.e., astrocytomas) was also included, and all preoperative imaging sequences were analyzed. Clinical variables including patient sex, age, and surgical blood loss were also included in the analysis. Images were acquired at both 1.5 and 3.0 T. The DSC images were acquired at a temporal resolution of either 1500 msec (3.0 T) or 2000 msec (1.5 T). In all cases, parameters including normalized cerebral blood volume (CBV) and transfer coefficient (kTrans) were calculated with region-of-interest analysis of enhancing tumor volume. The normalized CBV and kTrans data from the patient groups were analyzed with 1-way ANOVA, and post hoc statistical comparisons among groups were conducted with the Bonferroni adjustment. RESULTS Preoperative DSC imaging indicated mean (± SD) normalized CBVs of 5.7 ± 2.2 ml for WHO Grade I meningiomas of the meningoepithelial subtype (n = 12), 4.8 ± 1.8 ml for Grade IV astrocytomas (n = 54), and 12.3 ± 3.8 ml for Grade I meningiomas of the MM subtype (n = 12). The normalized CBV measured within the enhancing portion of the tumor was significantly higher in the MM subtype than in typical meningiomas and Grade IV astrocytomas (p < 0.001 for both). Preoperative DCE imaging indicated mean kTrans values of 0.49 ± 0.20 min-1 in Grade I meningiomas of the meningoepithelial subtype (n = 12), 0.27 ± 0.12 min-1 for Grade IV astrocytomas (n = 54), and 1.35 ± 0.74 min-1 for Grade I meningiomas of the MM subtype (n = 6). The kTrans was significantly higher in the MM variants than in the corresponding nonmicrocystic Grade 1 meningiomas and Grade IV astrocytomas (p < 0.001 for both). Intraoperative blood loss tended to increase with increased normalized CBV (R = 0.45, p = 0.085). CONCLUSIONS An enhancing cystic lesion with a normalized CBV greater than 10.3 ml or a kTrans greater than 0.88 min-1 should prompt radiologists and surgeons to consider the diagnosis of MM rather than traditional Grade I meningioma or high-grade glioma in planning surgical care. Higher normalized CBVs tend to be associated with increased intraoperative blood loss.
Asunto(s)
Neoplasias Encefálicas/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Glioma/diagnóstico por imagen , Imagen por Resonancia Magnética , Neoplasias Meníngeas/diagnóstico por imagen , Meningioma/diagnóstico por imagen , Neoplasias Encefálicas/patología , Estudios de Cohortes , Diagnóstico Diferencial , Femenino , Glioma/patología , Humanos , Interpretación de Imagen Asistida por Computador , Imagen por Resonancia Magnética/métodos , Masculino , Neoplasias Meníngeas/patología , Meningioma/patología , Persona de Mediana Edad , Clasificación del TumorRESUMEN
Poly(ADP-ribose) polymerase-1 (PARP-1) has become an important pharmacological target in the treatment of cancer due to its cellular role as a 'DNA-strand break sensor', which leads in part to resistance to some existing chemo- and radiological treatments. Inhibitors have now been developed which prevent PARP-1 from synthesizing poly(ADP-ribose) in response to DNA-breaks and potentiate the cytotoxicity of DNA damaging agents. However, with the recent discoveries of PARP-2, which has a similar DNA-damage dependent catalytic activity, and additional members containing the 'PARP catalytic' signature, the isoform selectivity and resultant pharmacological effects of existing inhibitors are brought into question. We present here the crystal structure of the catalytic fragment of murine PARP-2, at 2.8 A resolution, and compare this to the catalytic fragment of PARP-1, with an emphasis on providing a possible framework for rational drug design in order to develop future isoform-specific inhibitors.
Asunto(s)
Dominio Catalítico , Poli(ADP-Ribosa) Polimerasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Línea Celular , Pollos , Cristalización , Cristalografía por Rayos X , Daño del ADN , Diseño de Fármacos , Inhibidores Enzimáticos/química , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Conformación Proteica , Especificidad por SustratoRESUMEN
Standard tissue culture methods advise freezing cells in small aliquots (≤1 × 10(7) cells in 1 mL), and storing in liquid nitrogen. This is inconvenient for laboratories culturing large quantities of insect cells for recombinant baculovirus expression, owing to the length of time taken to produce large scale cultures from small aliquots of cells. Liquid nitrogen storage requires use of specialized cryovials, personal protective equipment and oxygen monitoring systems. This paper describes the long-term, large scale cryopreservation of 8 × 10(8) insect cells at -80 °C, using standard 50 mL conical tubes to contain a 40 mL cell suspension. Sf9, Sf21 and High 5 cells were recovered with a viability > 90 % after storage for one year under these conditions, which compared favorably with the viability of cells stored in liquid nitrogen for the same length of time. Addition of green fluorescent protein encoding baculovirus demonstrated that cells were "expression ready" immediately post thaw. Our method enables large scale cultures to be recovered rapidly from stocks cryopreserved at -80 °C, thus avoiding the inconvenience, hazards and expense associated with liquid nitrogen.
RESUMEN
Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/ultraestructura , Chaperoninas/química , Chaperoninas/ultraestructura , Quinasa 4 Dependiente de la Ciclina/química , Quinasa 4 Dependiente de la Ciclina/ultraestructura , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/ultraestructura , Proteínas de Ciclo Celular/aislamiento & purificación , Chaperoninas/aislamiento & purificación , Quinasa 4 Dependiente de la Ciclina/aislamiento & purificación , Proteínas HSP90 de Choque Térmico/aislamiento & purificación , Humanos , Microscopía Electrónica , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/ultraestructura , Unión ProteicaRESUMEN
The Cdc14 family of dual-specificity protein phosphatases (DSPs) is conserved within eukaryotes and functions to down-regulate mitotic Cdk activities, promoting cytokinesis and mitotic exit. We have integrated structural and kinetic analyses to define the molecular mechanism of the dephosphorylation reaction catalysed by Cdc14. The structure of Cdc14 illustrates a novel arrangement of two domains, each with a DSP-like fold, arranged in tandem. The C-terminal domain contains the conserved PTP motif of the catalytic site, whereas the N-terminal domain, which shares no sequence similarity with other DSPs, contributes to substrate specificity, and lacks catalytic activity. The catalytic site is located at the base of a pronounced surface channel formed by the interface of the two domains, and regions of both domains interact with the phosphopeptide substrate. Specificity for a pSer-Pro motif is mediated by a hydrophobic pocket that is capable of accommodating the apolar Pro(P+1) residue of the peptide. Our structural and kinetic data support a role for Cdc14 in the preferential dephosphorylation of proteins modified by proline-directed kinases.
Asunto(s)
Proteínas de Ciclo Celular/química , Fosfoproteínas Fosfatasas/metabolismo , Prolina/química , Proteínas Tirosina Fosfatasas/química , Proteínas de Saccharomyces cerevisiae/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/aislamiento & purificación , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The protein kinase Akt/PKB is stimulated by the phosphorylation of two regulatory residues, Thr 309 of the activation segment and Ser 474 of the hydrophobic motif (HM), that are structurally and functionally conserved within the AGC kinase family. To understand the mechanism of PKB regulation, we determined the crystal structures of activated kinase domains of PKB in complex with a GSK3beta-peptide substrate and an ATP analog. The activated state of the kinase was generated by phosphorylating Thr 309 using PDK1 and mimicking Ser 474 phosphorylation either with the S474D substitution or by replacing the HM of PKB with that of PIFtide, a potent mimic of a phosphorylated HM. Comparison with the inactive PKB structure indicates that the role of Ser 474 phosphorylation is to promote the engagement of the HM with the N-lobe of the kinase domain, promoting a disorder-to-order transition of the alphaC helix. The alphaC helix, by interacting with pThr 309, restructures and orders the activation segment, generating an active kinase conformation. Analysis of the interactions between PKB and the GSK3beta-peptide explains how PKB selects for protein substrates distinct from those of PKA.
Asunto(s)
Adenilil Imidodifosfato/química , Glucógeno Sintasa Quinasa 3/química , Modelos Moleculares , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Adenilil Imidodifosfato/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Interacciones Hidrofóbicas e Hidrofílicas , Sustancias Macromoleculares , Péptidos/química , Péptidos/metabolismo , Fosforilación , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por SustratoRESUMEN
Protein kinase B/Akt plays crucial roles in promoting cell survival and mediating insulin responses. The enzyme is stimulated by phosphorylation at two regulatory sites: Thr 309 of the activation segment and Ser 474 of the hydrophobic motif, a conserved feature of many AGC kinases. Analysis of the crystal structures of the unphosphorylated and Thr 309 phosphorylated states of the PKB kinase domain provides a molecular explanation for regulation by Ser 474 phosphorylation. Activation by Ser 474 phosphorylation occurs via a disorder to order transition of the alphaC helix with concomitant restructuring of the activation segment and reconfiguration of the kinase bilobal structure. These conformational changes are mediated by a phosphorylation-promoted interaction of the hydrophobic motif with a channel on the N-terminal lobe induced by the ordered alphaC helix and are mimicked by peptides corresponding to the hydrophobic motif of PKB and potently by the hydrophobic motif of PRK2.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Alineación de SecuenciaRESUMEN
Glycogen synthase kinase 3beta (GSK3beta) is a serine/threonine kinase involved in insulin, growth factor and Wnt signalling. In Wnt signalling, GSK3beta is recruited to a multiprotein complex via interaction with axin, where it hyperphosphorylates beta-catenin, marking it for ubiquitylation and destruction. We have now determined the crystal structure of GSK3beta in complex with a minimal GSK3beta-binding segment of axin, at 2.4 A resolution. The structure confirms the co-localization of the binding sites for axin and FRAT in the C-terminal domain of GSK3beta, but reveals significant differences in the interactions made by axin and FRAT, mediated by conformational plasticity of the 285-299 loop in GSK3beta. Detailed comparison of the axin and FRAT GSK3beta complexes allows the generation of highly specific mutations, which abrogate binding of one or the other. Quantitative analysis suggests that the interaction of GSK3beta with the axin scaffold enhances phosphorylation of beta-catenin by >20 000-fold.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Proteínas Portadoras , Glucógeno Sintasa Quinasa 3/química , Proteínas de Neoplasias , Estructura Terciaria de Proteína , Proteínas/química , Proteínas Represoras , Transducción de Señal/fisiología , Proteínas de Pez Cebra , Proteínas Adaptadoras Transductoras de Señales , Proteína de la Poliposis Adenomatosa del Colon/química , Proteína Axina , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Proteínas del Citoesqueleto/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Péptidos y Proteínas de Señalización Intracelular , Sustancias Macromoleculares , Modelos Moleculares , Estructura Molecular , Complejos Multiproteicos , Mutación , Fosforilación , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/metabolismo , Tirosina/metabolismo , Proteínas Wnt , beta CateninaRESUMEN
Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.