The brain isoform of a key ATP-regulating enzyme, creatine kinase, is a phosphoprotein.
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ABSTRACT:
SUBMITTER: Mahadevan LC
PROVIDER: S-EPMC1144154 | biostudies-other | 1984 Aug
REPOSITORIES: biostudies-other
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Project description:BackgroundCreatine Kinases (CK) catalyze the reversible transfer of high-energy phosphate groups between ATP and phosphocreatine, thereby playing a storage and distribution role in cellular energetics. Brain-type CK (CK-B) deficiency is coupled to loss of function in neural cell circuits, altered bone-remodeling by osteoclasts and complement-mediated phagocytotic activity of macrophages, processes sharing dependency on actomyosin dynamics.Methodology/principal findingsHere, we provide evidence for direct coupling between CK-B and actomyosin activities in cortical microdomains of astrocytes and fibroblasts during spreading and migration. CK-B transiently accumulates in membrane ruffles and ablation of CK-B activity affects spreading and migration performance. Complementation experiments in CK-B-deficient fibroblasts, using new strategies to force protein relocalization from cytosol to cortical sites at membranes, confirmed the contribution of compartmentalized CK-B to cell morphogenetic dynamics.Conclusion/significanceOur results provide evidence that local cytoskeletal dynamics during cell motility is coupled to on-site availability of ATP generated by CK-B.
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Project description:When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca(2+) regulation, and stress-response proteins, many of the most abundant bundle proteins that were identified by mass spectrometry were involved in ATP synthesis. After beta-actin, the cytosolic brain isoform of creatine kinase was the next most abundant bundle protein; at approximately 0.5 mM, creatine kinase is capable of maintaining high ATP levels despite 1 mM/s ATP consumption by the plasma-membrane Ca(2+)-ATPase. Consistent with this critical role in hair bundle function, the creatine kinase circuit is essential for high-sensitivity hearing as demonstrated by hearing loss in creatine kinase knockout mice.
| S-EPMC1839076 | biostudies-literature
Project description:Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and beta-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor-mediated, but not Fc-gammaR-mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.
| S-EPMC2265766 | biostudies-literature
Project description:Inositol hexakisphosphate kinases (IP6Ks) regulate various biological processes. IP6Ks convert IP6 to pyrophosphates such as diphosphoinositol pentakisphosphate (IP7) and bis-diphosphoinositol tetrakisphosphate (IP8). IP7 is produced in mammals by a family of inositol hexakisphosphate kinases, IP6K1, IP6K2, and IP6K3, which have distinct biological functions. The inositol hexakisphosphate kinase 2 (IP6K2) controls cellular apoptosis. To explore roles for IP6K2 in brain function, we elucidated its protein interactome in mouse brain revealing a robust association of IP6K2 with creatine kinase-B (CK-B), a key enzyme in energy homeostasis. Cerebella of IP6K2-deleted mice (IP6K2-knockout [KO]) produced less phosphocreatine and ATP and generated higher levels of reactive oxygen species and protein oxidative damage. In IP6K2-KO mice, mitochondrial dysfunction was associated with impaired expression of the cytochrome-c1 subunit of complex III of the electron transport chain. We reversed some of these effects by combined treatment with N-acetylcysteine and phosphocreatine. These findings establish a role for IP6K2-CK-B interaction in energy homeostasis associated with neuroprotection.
| S-EPMC8017945 | biostudies-literature
Project description:Calmodulin (CaM) is a ubiquitous Ca2+ sensing protein that binds to and modulates numerous target proteins and enzymes during cellular signaling processes. A large number of CaM-target complexes have been identified and structurally characterized, revealing a wide diversity of CaM-binding modes. A newly identified target is creatine kinase (CK), a central enzyme in cellular energy homeostasis. This study reports two high-resolution X-ray structures, determined to 1.24 Å and 1.43 Å resolution, of calmodulin in complex with peptides from human brain and muscle CK, respectively. Both complexes adopt a rare extended binding mode with an observed stoichiometry of 1:2 CaM:peptide, confirmed by isothermal titration calorimetry, suggesting that each CaM domain independently binds one CK peptide in a Ca2+-depended manner. While the overall binding mode is similar between the structures with muscle or brain-type CK peptides, the most significant difference is the opposite binding orientation of the peptides in the N-terminal domain. This may extrapolate into distinct binding modes and regulation of the full-length CK isoforms. The structural insights gained in this study strengthen the link between cellular energy homeostasis and Ca2+-mediated cell signaling and may shed light on ways by which cells can 'fine tune' their energy levels to match the spatial and temporal demands.
| S-EPMC8244255 | biostudies-literature
Project description:³¹P magnetization saturation transfer (MST) experiment is the most widely used method to study ATP metabolism kinetics. However, its lengthy data acquisition time greatly limits the wide biomedical applications in vivo, especially for studies requiring high spatial and temporal resolutions.We aimed to develop a novel superfast MST method that can accurately quantify ATP production rate constants (k(f)) through creatine kinase (CK) or ATP synthase (ATPase) with 2 spectra.The T?(nom) (T? nominal) method uses a correction factor to compensate the partially relaxed MST experiments, thus allowing measurement of enzyme kinetics with an arbitrary repetition time and flip angle, which consequently reduces the data acquisition time of a transmurally differentiated CK k(f) measurement by 91% as compared with the conventional method with spatial localization. The novel T?(nom) method is validated theoretically with numeric simulation, and further verified with in vivo swine hearts, as well as CK and ATPase activities in rat brain at 9.4 Tesla. Importantly, the in vivo data from swine hearts demonstrate, for the first time, that within an observation window of 30 minutes, the inhibition of CK activity by iodoacetamide does not limit left ventricular chamber contractile function.A novel MST method for superfast examination of enzyme kinetics in vivo has been developed and verified theoretically and experimentally. In the in vivo normal heart, redundant multiple supporting systems of myocardial ATP production, transportation, and utilization exist, such that inhibition of one mechanism does not impair the normal left ventricular contractile performance.
| S-EPMC3090083 | biostudies-literature
Project description:BackgroundObesity is strongly associated with exercise intolerance and the development of heart failure. Whereas myocardial energetics and diastolic function are impaired in obesity, systolic function is usually preserved. This suggests that the rate of ATP delivery is maintained, but this has never been explored in human obesity. We hypothesized that ATP transfer rate through creatine kinase (CK) (kfCKrest) would be increased, compensating for depleted energy stores (phosphocreatine/ATP), but potentially limiting greater ATP delivery during increased workload. We hypothesized that these changes would normalize with weight loss.MethodsWe recruited 80 volunteers (35 controls [body mass index 24±3 kg/m2], 45 obese [body mass index 35±5 kg/m2]) without coexisting cardiovascular disease. Participants underwent body composition analysis, magnetic resonance imaging of abdominal, liver, and myocardial fat content, left ventricular function, and 31P magnetic resonance spectroscopy to assess phosphocreatine/ATP and CK kinetics, at rest and during dobutamine stress. Obese volunteers were assigned to a dietary weight loss intervention, before reexamination.ResultsAt rest, although myocardial phosphocreatine/ATP was 14% lower in obesity (1.9±0.3 versus 2.2±0.2, P<0.001), kfCkrest was 33% higher (0.23±0.07 s-1 versus 0.16±0.08 s-1, P=0.002), yielding no difference in overall resting ATP delivery (obese 2.5±0.9 µmol·g-1·s-1 versus control 2.2±1.1 µmol·g-1·s-1, P=0.232). In controls, increasing cardiac workload led to an increase in both kfCK (+86%, P<0.001) and ATP delivery (+80%, P<0.001). However, in obesity, similar stress led to no significant increase in either kfCK (P=0.117) or ATP delivery (P=0.608). This was accompanied by reduced systolic augmentation (absolute increase in left ventricular ejection fraction, obese +16±7% versus control +21±4%, P=0.031). Successful weight loss (-11±5% body weight) was associated with improvement of these energetic changes such that there was no significant difference in comparison with controls.ConclusionsIn the obese resting heart, the myocardial CK reaction rate is increased, maintaining ATP delivery despite reduced phosphocreatine/ATP. During increased workload, although the nonobese heart increases ATP delivery through CK, the obese heart does not; this is associated with reduced systolic augmentation and exercise tolerance. Weight loss reverses these energetic changes. This highlights myocardial energy delivery through CK as a potential therapeutic target to improve symptoms in obesity-related heart disease, and a fascinating modifiable pathway involved in the progression to heart failure, as well.
| S-EPMC7144750 | biostudies-literature
Project description:Protection against inhibition of creatine kinase by iodoacetamide is measured by the decrease in the rate constant for the inhibition reaction; A mixture of purified substrates at equilibrium protects quite strongly when all the components of the mixture are nearly saturating. The protection by substrates 'working' in the forward direction only (from creatine and MgATP) was measured by carrying out the experiment rapidly at low concentrations of the enzyme; by varying the concentration of substrate it was found that the amount of protection when the substrates of the forward reaction are saturating is about 80% (100% protection would imply a value of zero for the rate constant of the inhibition reaction). The effects of Ca2+ and Mg2+ are compared. It is already known that the complex creatine-NO3--MgADP, which is considered to be either a transition-state analogue or an analogue of an intermediate in the reaction pathway, protects fully against iodoacetamide, whereas creatine and MgADP alone, or together without NO3-, do not protect. This suggests that the degree of protection by the working enzyme represents the proportion of enzyme molecules that have a conformation complementary to a creatine-PO3-MgADP intermediate.
| S-EPMC1163814 | biostudies-other
Project description:The dihydroxyacetone kinase (DhaK) of Escherichia coli consists of three soluble protein subunits. DhaK (YcgT; 39.5 kDa) and DhaL (YcgS; 22.6 kDa) are similar to the N- and C-terminal halves of the ATP-dependent DhaK ubiquitous in bacteria, animals and plants. The homodimeric DhaM (YcgC; 51.6 kDa) consists of three domains. The N-terminal dimerization domain has the same fold as the IIA domain (PDB code 1PDO) of the mannose transporter of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The middle domain is similar to HPr and the C-terminus is similar to the N-terminal domain of enzyme I (EI) of the PTS. DhaM is phosphorylated three times by phosphoenolpyruvate in an EI- and HPr-dependent reaction. DhaK and DhaL are not phosphorylated. The IIA domain of DhaM, instead of ATP, is the phosphoryl donor to dihydroxyacetone (Dha). Unlike the carbohydrate-specific transporters of the PTS, DhaK, DhaL and DhaM have no transport activity.
| S-EPMC125457 | biostudies-literature
Project description:Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.
| S-EPMC8448711 | biostudies-literature