RESUMO
Ca2+/calmodulin-dependent protein kinase kinases (CaMKKα and ß) are regulatory kinases for multiple downstream kinases, including CaMKI, CaMKIV, PKB/Akt, and AMP-activated protein kinase (AMPK) through phosphorylation of each activation-loop Thr residue. In this report, we biochemically characterize the oligomeric structure of CaMKK isoforms through a heterologous expression system using COS-7 cells. Oligomerization of CaMKK isoforms was readily observed by treating CaMKK transfected cells with cell membrane permeable crosslinkers. In addition, His-tagged CaMKKα (His-CaMKKα) pulled down with FLAG-tagged CaMKKα (FLAG-CaMKKα) in transfected cells. The oligomerization of CaMKKα was confirmed by the fact that GST-CaMKKα/His-CaMKKα complex from transiently expressed COS-7 cells extracts was purified to near homogeneity by the sequential chromatography using glutathione-sepharose/Ni-sepharose and was observed in a Ca2+/CaM-independent manner by reciprocal pulldown assay, suggesting the direct interaction between monomeric CaMKKα. Furthermore, the His-CaMKKα kinase-dead mutant (D293A) complexed with FLAG-CaMKKα exhibited significant CaMKK activity, indicating the active CaMKKα multimeric complex. Collectively, these results suggest that CaMKKα can self-associate in the cells, constituting a catalytically active oligomer that might be important for the efficient activation of CaMKK-mediated intracellular signaling.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/química , Glutationa Transferase/química , Proteínas Recombinantes de Fusão/química , Animais , Sítios de Ligação , Células COS , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Chlorocebus aethiops , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Fosforilação , Ligação Proteica , Multimerização Proteica , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Locomotive syndrome (LS) includes disorders of the musculoskeletal system and is a high-risk condition that requires nursing care. We included an examination of locomotive function in specific health checkups to investigate the relationship between LS risk and the muscle strengths. The purpose of this study was to determine the distribution of LS and the relationship between LS and metabolic syndrome (MetS). METHODS: Among 2695 participants who undertook a specific health checkup, 790 received a locomotive examination (302 men and 488 women; mean age, 65.9 years). Data for MetS components were measured in the specific health checkup. Data about the locomotive examination were obtained from five tests: the two-step test, stand-up test, 25-question Geriatric Locomotive Function Scale, and measurement of lower limb muscular strength and grip strength. RESULTS: The MetS components did not differ according to LS risk level in men. In women, body weight, body mass index, and abdominal circumference were significantly lower in the non-LS group than in the LS risk level 1 or 2 groups. The ratio of lower limb muscular strength to body weight differed significantly between all risk groups in men and women. In women, lower limb muscular strength was significantly higher in those at risk of both LS and MetS (double-risk group) than in the LS-only group. In women, the ratio of lower limb muscular strength to body weight was significantly lower in the double-risk group than in the LS-only group. CONCLUSIONS: Adding a locomotive examination to the specific health checkup may be useful for identifying people at risk of LS or MetS. The prevalence rates of LS and MetS correlate positively in women. The ratio of lower limb muscular strength to body weight might be a better index of locomotor dysfunction than lower limb muscular strength alone.
Assuntos
Locomoção/fisiologia , Síndrome Metabólica/complicações , Síndrome Metabólica/fisiopatologia , Limitação da Mobilidade , Força Muscular/fisiologia , Equilíbrio Postural/fisiologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , SíndromeRESUMO
A taste sensor that uses lipid/polymer membranes can evaluate aftertastes felt by humans using Change in membrane Potential caused by Adsorption (CPA) measurements. The sensor membrane for evaluating bitterness, which is caused by acidic bitter substances such as iso-alpha acid contained in beer, needs an immersion process in monosodium glutamate (MSG) solution, called "MSG preconditioning". However, what happens to the lipid/polymer membrane during MSG preconditioning is not clear. Therefore, we carried out three experiments to investigate the changes in the lipid/polymer membrane caused by the MSG preconditioning, i.e., measurements of the taste sensor, measurements of the amount of the bitterness substance adsorbed onto the membrane and measurements of the contact angle of the membrane surface. The CPA values increased as the preconditioning process progressed, and became stable after 3 d of preconditioning. The response potentials to the reference solution showed the same tendency of the CPA value change during the preconditioning period. The contact angle of the lipid/polymer membrane surface decreased after 7 d of MSG preconditioning; in short, the surface of the lipid/polymer membrane became hydrophilic during MSG preconditioning. The amount of adsorbed iso-alpha acid was increased until 5 d preconditioning, and then it decreased. In this study, we revealed that the CPA values increased with the progress of MSG preconditioning in spite of the decrease of the amount of iso-alpha acid adsorbed onto the lipid/polymer membrane, and it was indicated that the CPA values increase because the sensor sensitivity was improved by the MSG preconditioning.
RESUMO
A taste sensor using lipid-polymer membranes has been developed to evaluate the taste of foods, beverages and medicines. The response of the taste sensor, measured as a change in the membrane potential caused by adsorption (CPA), corresponds to the aftertaste felt by humans. The relationships between the CPA value and the amount of adsorbed taste substances, quinine and iso-α acid (bitterness), and tannic acid (astringency), have been studied so far. However, that of epigallocatechin gallate (EGCg) has not been clarified, although EGCg is abundantly present in green tea as one of its astringent substances. This study aimed at clarifying the response of the taste sensor to EGCg and its relationship with the amount of EGCg adsorbed onto lipid-polymer membranes. The lipid concentration dependence of the CPA value was similar to that of the amount of adsorbed EGCg, indicating a high correlation between the CPA value and the amount of adsorbed EGCg. The CPA value increased with increasing amount of adsorbed EGCg; however, the CPA value showed a tendency of leveling off when the amount of adsorbed EGCg further increased.
Assuntos
Técnicas Biossensoriais , Catequina/análogos & derivados , Membranas Artificiais , Paladar/fisiologia , Adsorção , Catequina/química , Catequina/isolamento & purificação , Humanos , Lipídeos/química , Polímeros/químicaRESUMO
Calcium/calmodulin-dependent protein kinase kinases (CaMKKs) activate CaMKI, CaMKIV, protein kinase B/Akt, and AMP-activated protein kinase (AMPK) by phosphorylating Thr residues in activation loops to mediate various Ca2+ -signaling pathways. Mammalian cells expressing CaMKKα and CaMKKß lacking Arg/Pro-rich insert domain (RP-domain) sequences showed impaired phosphorylation of AMPKα, CaMKIα, and CaMKIV, whereas the autophosphorylation activities of CaMKK mutants remained intact and were similar to those of wild-type CaMKKs. Liver kinase B1 (LKB1, an AMPK kinase) complexed with STRAD and MO25 and was unable to phosphorylate CaMKIα and CaMKIV; however, mutant LKB1 with the RP-domain sequences of CaMKKα and CaMKKß inserted between kinase subdomains II and III acquired CaMKIα and CaMKIV phosphorylating activity in vitro and in transfected cultured cells. Furthermore, ionomycin-induced phosphorylation of hemagglutinin (HA)-CaMKIα at Thr177, HA-CaMKIV at Thr196, and HA-AMPKα at Thr172 in transfected cells was significantly suppressed by cotransfection of kinase-dead mutants of CaMKK isoforms, but these dominant-negative effects were abrogated with RP-deletion mutants, suggesting that sequestration of substrate kinases by loss-of-function CaMKK mutants requires the RP-domain. This was confirmed by pulldown experiments that showed that dominant-negative mutants of CaMKKα and CaMKKß interact with target kinases but not RP-deletion mutants. Taken together, these results clearly indicate that both CaMKK isoforms require the RP-domain to recognize downstream kinases to interact with and phosphorylate Thr residues in their activation loops. Thus, the RP-domain may be a promising target for specific CaMKK inhibitors.