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1.
Nat Immunol ; 25(8): 1355-1366, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39014161

RESUMEN

Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity; however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell antigen receptor (γδTCR) in complex with BTN2A1 revealed that BTN2A1 engages the side of the γδTCR, leaving the apical TCR surface bioavailable. We reveal that a second γδTCR ligand co-engages γδTCR via binding to this accessible apical surface in a BTN3A1-dependent manner. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1-BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to mediate the γδTCR interaction; indeed, locking BTN2A1 and BTN3A1 together abrogated their interaction with γδTCR, supporting a model wherein the two γδTCR ligand-binding sites depend on accessibility to cryptic BTN epitopes. Our findings reveal a new paradigm in immune activation, whereby γδTCRs sense dual epitopes on BTN complexes.


Asunto(s)
Butirofilinas , Receptores de Antígenos de Linfocitos T gamma-delta , Butirofilinas/metabolismo , Butirofilinas/inmunología , Butirofilinas/química , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Humanos , Unión Proteica , Multimerización de Proteína , Antígenos CD/metabolismo , Antígenos CD/inmunología , Antígenos CD/química , Linfocitos T/inmunología , Linfocitos T/metabolismo , Cristalografía por Rayos X , Activación de Linfocitos/inmunología , Modelos Moleculares , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismo
2.
Mol Cell ; 30(6): 767-78, 2008 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-18570878

RESUMEN

Forkhead-associated (FHA) domains recognize phosphothreonines, and SQ/TQ cluster domains (SCDs) contain concentrated phosphorylation sites for ATM/ATR-like DNA-damage-response kinases. The Rad53-SCD1 has dual functions in regulating the activation of the Rad53-Dun1 checkpoint kinase cascade but with unknown molecular mechanisms. Here we present structural, biochemical, and genetic evidence that Dun1-FHA possesses an unprecedented diphosphothreonine-binding specificity. The Dun1-FHA has >100-fold increased affinity for diphosphorylated relative to monophosphorylated Rad53-SCD1 due to the presence of two separate phosphothreonine-binding pockets. In vivo, any single threonine of Rad53-SCD1 is sufficient for Rad53 activation and RAD53-dependent survival of DNA damage, but two adjacent phosphothreonines in the Rad53-SCD1 and two phosphothreonine-binding sites in the Dun1-FHA are necessary for Dun1 activation and DUN1-dependent transcriptional responses to DNA damage. The results uncover a phospho-counting mechanism that regulates the specificity of SCD, and provide mechanistic insight into a role of multisite phosphorylation in DNA-damage signaling.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fosfotreonina/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Sitios de Unión , Proteínas de Ciclo Celular/química , Quinasa de Punto de Control 2 , Daño del ADN , ADN de Hongos/genética , Activación Enzimática , Cinética , Ligandos , Fosfotreonina/química , Unión Proteica , Proteínas Quinasas/química , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Sensibilidad y Especificidad
3.
PLoS Genet ; 6(10): e1001170, 2010 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-20975950

RESUMEN

Zn²(+)-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn²(+)-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis.


Asunto(s)
Proteínas Portadoras/fisiología , Reparación del ADN/fisiología , Pulmón/embriología , Proteínas Nucleares/fisiología , Organogénesis/fisiología , Animales , Western Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Senescencia Celular , Daño del ADN , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Genotipo , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxidantes/farmacología , Factores de Tiempo , Tráquea/embriología , Factores de Transcripción , Rayos Ultravioleta
4.
J Invest Dermatol ; 142(4): 1103-1113.e11, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-34537191

RESUMEN

Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (ßc) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit ßc, a property that has made ßc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of ßc has precluded testing of inhibitors of human ßc in mouse models. To overcome this problem, we developed a human ßc receptor transgenic mouse strain with a hematopoietic cell‒specific expression of human ßc instead of mouse ßc. Human ßc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3-/- mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking anti-human ßc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human ßc receptor transgenic mouse as a unique platform to test the inhibitors of ßc in vivo.


Asunto(s)
Dermatitis por Contacto , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Animales , Citocinas , Eosinófilos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Ratones , Ratones Transgénicos
5.
Science ; 367(6478)2020 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-31919129

RESUMEN

Gamma delta (γδ) T cells are essential to protective immunity. In humans, most γδ T cells express Vγ9Vδ2+ T cell receptors (TCRs) that respond to phosphoantigens (pAgs) produced by cellular pathogens and overexpressed by cancers. However, the molecular targets recognized by these γδTCRs are unknown. Here, we identify butyrophilin 2A1 (BTN2A1) as a key ligand that binds to the Vγ9+ TCR γ chain. BTN2A1 associates with another butyrophilin, BTN3A1, and these act together to initiate responses to pAg. Furthermore, binding of a second ligand, possibly BTN3A1, to a separate TCR domain incorporating Vδ2 is also required. This distinctive mode of Ag-dependent T cell activation advances our understanding of diseases involving pAg recognition and creates opportunities for the development of γδ T cell-based immunotherapies.


Asunto(s)
Antígenos de Neoplasias/inmunología , Butirofilinas/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Antígenos CD/química , Antígenos CD/inmunología , Butirofilinas/química , Butirofilinas/genética , Línea Celular Tumoral , Humanos , Ligandos , Activación de Linfocitos , Fosforilación , Dominios Proteicos , Multimerización de Proteína
6.
Genetics ; 169(1): 65-75, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15466434

RESUMEN

DNA damage checkpoints regulate gene expression at the transcriptional and post-transcriptional level. Some components of the yeast Ccr4-Not complex, which regulates transcription as well as transcript turnover, have previously been linked to DNA damage responses, but it is unclear if this involves transcriptional or post-transcriptional functions. Here we show that CCR4 and CAF1, which together encode the major cytoplasmic mRNA deadenylase complex, have complex genetic interactions with the checkpoint genes DUN1, MRC1, RAD9, and RAD17 in response to DNA-damaging agents hydroxyurea (HU) and methylmethane sulfonate (MMS). The exonuclease-inactivating ccr4-1 point mutation mimics ccr4Delta phenotypes, including synthetic HU hypersensitivity with dun1Delta, demonstrating that Ccr4-Not mRNA deadenylase activity is required for DNA damage responses. However, ccr4Delta and caf1Delta DNA damage phenotypes and genetic interactions with checkpoint genes are not identical, and deletions of some Not components that are believed to predominantly function at the transcriptional level rather than mRNA turnover, e.g., not5Delta, also lead to increased DNA damage sensitivity and synthetic HU hypersensitivity with dun1Delta. Taken together, our data thus suggest that both transcriptional and post-transcriptional functions of the Ccr4-Not complex contribute to the DNA damage response affecting gene expression in a complex manner.


Asunto(s)
Daño del ADN , Ribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Hidroxiurea/farmacología , Metilmetanosulfonato/farmacología , Mutágenos/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fenotipo , Mutación Puntual , Ribonucleasas/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética
7.
NPJ Vaccines ; 1: 16022, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-29263860

RESUMEN

Porphyromonas gingivalis infected mice with an established P. gingivalis-specific inflammatory immune response were protected from developing alveolar bone resorption by therapeutic vaccination with a chimera (KAS2-A1) immunogen targeting the major virulence factors of the bacterium, the gingipain proteinases. Protection was characterised by an antigen-specific IgG1 isotype antibody and Th2 cell response. Adoptive transfer of KAS2-A1-specific IgG1 or IgG2 expressing B cells confirmed that IgG1-mediated protection. Furthermore, parenteral or intraoral administration of KAS2-A1-specific polyclonal antibodies protected against the development of P. gingivalis-induced bone resorption. The KAS2-A1-specific antibodies neutralised the gingipains by inhibiting: proteolytic activity, binding to host cells/proteins and co-aggregation with other periodontal bacteria. Combining key gingipain sequences into a chimera vaccine produced an effective therapeutic intervention that protected against P. gingivalis-induced periodontitis.

8.
G3 (Bethesda) ; 3(10): 1649-59, 2013 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-23893744

RESUMEN

During its natural life cycle, budding yeast (Saccharomyces cerevisiae) has to adapt to drastically changing environments, but how environmental-sensing pathways are linked to adaptive gene expression changes remains incompletely understood. Here, we describe two closely related yeast hEST1A-B (SMG5-6)-like proteins termed Esl1 and Esl2 that contain a 14-3-3-like domain and a putative PilT N-terminus ribonuclease domain. We found that, unlike their metazoan orthologs, Esl1 and Esl2 were not involved in nonsense-mediated mRNA decay or telomere maintenance pathways. However, in genome-wide expression array analyses, absence of Esl1 and Esl2 led to more than two-fold deregulation of ∼50 transcripts, most of which were expressed inversely to the appropriate metabolic response to environmental nutrient supply; for instance, normally glucose-repressed genes were derepressed in esl1Δ esl2Δ double mutants during growth in a high-glucose environment. Likewise, in a genome-wide synthetic gene array screen, esl1Δ esl2Δ double mutants were synthetic sick with null mutations for Rim8 and Dfg16, which form the environmental-sensing complex of the Rim101 pH response gene expression pathway. Overall, these results suggest that Esl1 and Esl2 contribute to the regulation of adaptive gene expression responses of environmental sensing pathways.


Asunto(s)
Proteínas Portadoras/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telomerasa/metabolismo , Adaptación Fisiológica/genética , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Eliminación de Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Telomerasa/genética , Transcripción Genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo
9.
Mol Cell Biol ; 33(16): 3202-13, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23754745

RESUMEN

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2 , ADN de Hongos/genética , Activación Enzimática , Eliminación de Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteolisis , Fase S , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
10.
EMBO Rep ; 8(9): 851-7, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17721446

RESUMEN

Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage-dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell-cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this interaction results in in vivo phenotypes that are similar to those caused by an H2A phosphorylation site mutation. Remarkably, similar checkpoint defects are also caused by a Rad9 Tudor domain mutation that impairs Rad9 chromatin association already in undamaged cells. These findings indicate that constitutive Tudor domain-mediated and damage-specific BRCT domain-phospho-H2A-dependent interactions of Rad9 with chromatin cooperate to establish G1 checkpoint arrest.


Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Fase G1 , Histonas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal
11.
Biochem Biophys Res Commun ; 357(3): 800-3, 2007 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-17442269

RESUMEN

Bleomycins are small glycopeptide cancer chemotherapeutics that give rise to 3'-modified DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, DSBs are predominantly repaired by RAD52-dependent homologous recombination (HR) with some support by Yku70/Yku80 (KU)-dependent pathways. The main DSB repair function of KU is believed to be as part of the non-homologous end-joining (NHEJ) pathway, but KU also functions in a "chromosome healing" pathway that seals DSBs by de novo telomere addition. We report here that rad52Deltayku70Delta double mutants are considerably more bleomycin hypersensitive than rad52Deltalig4Delta cells that lack the NHEJ-specific DNA ligase 4. Moreover, the telomere-specific KU mutation yku80-135i also dramatically increases rad52Delta bleomycin hypersensitivity, almost to the level of rad52Deltayku80Delta. The results indicate that telomere-specific functions of KU play a more prominent role in the repair of bleomycin-induced damage than its NHEJ functions, which could have important clinical implications for bleomycin-based combination chemotherapies.


Asunto(s)
Antígenos Nucleares/fisiología , Bleomicina/toxicidad , Daño del ADN , Reparación del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Telómero/metabolismo , Antibióticos Antineoplásicos/toxicidad , Antígenos Nucleares/genética , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Ligasas/fisiología , Reparación del ADN/genética , ADN de Hongos/efectos de los fármacos , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Autoantígeno Ku , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/genética , Mutación , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/fisiología , Recombinación Genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Telómero/genética
12.
J Biol Chem ; 277(25): 22469-74, 2002 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-11953437

RESUMEN

The yeast Dun1 kinase has complex checkpoint functions including DNA damage-dependent cell cycle arrest in G(2)/M, transcriptional induction of repair genes, and regulation of postreplicative DNA repair pathways. Here we report that the Dun1 forkhead-associated domain interacts with the Pan3 subunit of the poly(A)-nuclease complex and that dun1pan2 and dun1pan3 double mutants are dramatically hypersensitive to replicational stress. This phenotype was independent of the function of Dun1 in regulating deoxyribonucleotide levels as it was also observed in strains lacking the ribonucleotide reductase inhibitor Sml1. dun1pan2 mutants initially arrested normally in response to replication blocks but died in the presence of persistent replication blocks with considerably delayed kinetics compared with mutants lacking the Rad53 kinase, indicating that the double mutation does not compromise the intra-S phase checkpoint. Interestingly, the RAD5 gene involved in error-free postreplication repair pathways was specifically up-regulated in dun1pan2 double mutants. Moreover, inducible overexpression of RAD5 mimicked the double mutant phenotype by hypersensitizing dun1 mutants to replication blocks. The data indicate that Dun1 and Pan2-Pan3 cooperate to regulate the stoichiometry and thereby the activity of postreplication repair complexes, suggesting that posttranscriptional mechanisms complement the transcriptional response in the regulation of gene expression by checkpoint signaling pathways in Saccharomyces cerevisiae.


Asunto(s)
Adenosina Trifosfatasas , Proteínas de Ciclo Celular , Exorribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinasas/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Saccharomyces cerevisiae , Northern Blotting , Núcleo Celular/metabolismo , Supervivencia Celular , Quinasa de Punto de Control 2 , Citoplasma/metabolismo , ADN Helicasas , Relación Dosis-Respuesta a Droga , Proteínas Fúngicas/genética , Hidroxiurea/farmacología , Cinética , Metilmetanosulfonato/farmacología , Modelos Genéticos , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , Regulación hacia Arriba
13.
IUBMB Life ; 55(1): 23-7, 2003 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12716058

RESUMEN

Forkhead-associated (FHA) domains are present in >200 diverse proteins in all phyla from bacteria to mammals and seem to be particularly prevalent in proteins with cell cycle control functions. Recent work from several laboratories has considerably improved our understanding of the structure and function of these domains that were virtually unknown a few years ago, and the first disease associations of FHA domains have now emerged. FHA domains form 11-stranded beta-sandwiches that contain some 100-180 amino acid residues with a high degree of sequence diversity. FHA domains act as phosphorylation-dependent protein-protein interaction modules that preferentially bind to phospho-threonine residues in their targets. Interestingly, point mutations in the human CHK2 gene that lead to single-residue amino acid substitutions in the FHA domain of this cell cycle checkpoint kinase have been found to cause a subset of cases of the Li-Fraumeni multi-cancer syndrome.


Asunto(s)
Fosfotreonina/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Ciclo Celular/fisiología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia
14.
Biochem Biophys Res Commun ; 293(3): 892-8, 2002 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-12051742

RESUMEN

The AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase important for the responses to metabolic stress. It consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, and is regulated both by the allosteric action of AMP and by phosphorylation of the alpha and beta subunits catalyzed by AMPKK(s) and autophosphorylation. The Thr172 site on the alpha subunit has been previously characterized as an activating phosphorylation site. Using bacterially expressed AMPK alpha1 subunit proteins, we have explored the role of Thr172-directed AMPKKs in alpha subunit regulation. Recombinant alpha1 subunit proteins, representing the N-terminus, have been expressed as maltose binding protein (MBP) 6x His fusion proteins and purified to homogeneity by Ni(2+) chromatography. Both wild-type alpha1(1-312) and alpha1(1-312)T172D are inactive when expressed in bacteria, but the former can be fully phosphorylated (1 mol/mol) on Thr172 and activated by a surrogate AMPKK, CaMKKbeta. The corresponding AMPKalpha1(1-392), an alpha construct containing its autoinhibitory sequence, can be similarly phosphorylated, but it remains inactive. In an insulinoma cell line, either low glucose or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) treatment leads to activation and T172 phosphorylation of endogenous AMPK. Under the same conditions of cell incubation, we have identified an AMPKK activity that both phosphorylates and activates the recombinant alpha1(1-312), but this Thr172-directed AMPKK activity is unaltered by low glucose or AICAR, indicating that it is constitutively active.


Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Proteínas Quinasas/análisis , Quinasas de la Proteína-Quinasa Activada por el AMP , Aminoimidazol Carboxamida/farmacología , Animales , Glucosa/farmacología , Fosforilación , Fosfotreonina/metabolismo , Subunidades de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleótidos/farmacología , Células Tumorales Cultivadas
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