Choline Kinase

The effect of expression of the Harvey-ras oncogene on phosphatidylcholine metabolism in C3H10T1/2 mouse fibroblast cells was examined. There were multiple changes in the CDP-choline pathway for phosphatidylcholine biosynthesis in the ras-expressing cells. The activity of the first enzyme in the pathway, choline kinase, was stimulated 1.9-fold, while the activity of the second enzyme, CTP:phosphocholine cytidylyltransferase, was decreased by one-half. High levels of intracellular phosphocholine measured in the ras cells were consistent with the altered activities of choline kinase and cytidylyltransferase. The overall rate of phosphatidylcholine synthesis appeared to be increased because the turnover rate of phosphocholine from the intracellular pool was higher in the ras-transfected cells. There also appeared to be an increased rate of phosphatidylcholine degradation in ras-expressing C3H10T1/2 cells. Very high levels of glycerophosphocholine (6-fold increased over control cells) s...

Morphine tolerance remains a challenge in the management of chronic pain in the clinic. As shown in our previous study, the dopamine D2 receptor (D2DR) expressed in spinal cord neurons might be involved in morphine tolerance, but the underlying mechanisms remain to be elucidated. In the present study, selective spinal D2DR blockade attenuated morphine tolerance in mice by inhibiting phosphatidylinositol 3 kinase (PI3K)/serine-threonine kinase (Akt)-mitogen activated protein kinase (MAPK) signaling in a μ opioid receptor (MOR)-dependent manner. Levocorydalmine (l-CDL), which exhibited micromolar affinity for D2DR in D2/CHO-K1 cell lines in this report and effectively alleviated bone cancer pain in our previous study, attenuated morphine tolerance in rats with chronic bone cancer pain at nonanalgesic doses. Furthermore, the intrathecal administration of l-CDL obviously attenuated morphine tolerance, and the effect was reversed by a D2DR agonist in mice. Spinal D2DR inhibition and l-CDL also inhibited tolerance induced by the MOR agonist DAMGO. l-CDL and a D2DR small interfering RNA (siRNA) decreased the increase in levels of phosphorylated Akt and MAPK in the spinal cord; these changes were abolished by a PI3K inhibitor. In addition, the activated Akt and MAPK proteins in mice exhibiting morphine tolerance were inhibited by a MOR antagonist. Intrathecal administration of a PI3K inhibitor also attenuated DAMGO-induced tolerance. Based on these results, l-CDL antagonized spinal D2DR to attenuate morphine tolerance by inhibiting PI3K/Akt-dependent MAPK phosphorylation through MOR. These findings provide insights into a more versatile treatment for morphine tolerance.

Purpose: Low concentrations of citrate and high concentrations of choline-containing compounds (ChoCC) are metabolic characteristics observed by magnetic resonance spectroscopy of prostate cancer tissue. The objective was to investigate the gene expression changes underlying these metabolic aberrations to find regulatory genes with potential for targeted therapies. Experimental design: Fresh frozen samples (n ¼ 133) from 41 patients undergoing radical prostatectomy were included. Histopathologic evaluation was carried out for each sample before a metabolic profile was obtained with high-resolution magic angle spinning (HR-MAS) spectroscopy. Following the HR-MAS, RNA was extracted from the same sample and quality controlled before carrying out microarray gene expression profiling. A partial least square statistical model was used to integrate the data sets to identify genes whose expression show significant covariance with citrate and ChoCC levels. Results: Samples were classified as benign, n ¼ 35; cancer of low grade (Gleason score 6), n ¼ 24; intermediate grade (Gleason score 7), n ¼ 41; or high grade (Gleason score !8), n ¼ 33. RNA quality was high with a mean RNA Integrity Number score of 9.1 (SD 1.2). Gene products predicting significantly a reduced citrate level were acetyl citrate lyase (ACLY, P ¼ 0.003) and m-aconitase (ACON, P < 0.001). The two genes whose expression most closely accompanied the increase in ChoCC were those of phospholipase A2 group VII (PLA2G7, P < 0.001) and choline kinase a (CHKA, P ¼ 0.002). Conclusions: By integrating histologic, transcriptomic, and metabolic data, our study has contributed to an expanded understanding of the mechanisms underlying aberrant citrate and ChoCC levels in prostate cancer. Clin Cancer Res; 18(12); 3261-9. Ó2012 AACR.

Choline kinase in mice is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneously occurring genomic deletion in murine Chkb results in neonatal bone deformity and hindlimb muscular dystrophy. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, causes hindlimb muscular dystrophy. The biosynthesis of phosphatidylcholine (PC) is impaired in the hindlimbs of Chkb −/− mice, with an accumulation of choline and decreased amount of phosphocholine. The activity of CTP:phosphocholine cytidylyltransferase is also decreased in the hindlimb muscle of mutant mice. Concomitantly, the activities of PC phospholipase C and phospholipase A 2 are increased. The mitochondria in Chkb −/− mice are abnormally large and exhibit decreased inner membrane potential. Despite the muscular dystrophy in Chkb −/− mice, we observed increased expression of insulin like growth factor 1 and proliferating cell nuclear antigen. However, regeneration of hindlimb muscles of Chkb −/− mice was impaired when challenged with cardiotoxin. Injection of CDP-choline increased PC content of hindlimb muscle and decreased creatine kinase activity in plasma of Chkb −/− mice. We conclude that the hindlimb muscular dystrophy in Chkb −/− mice is due to attenuated PC biosynthesis and enhanced catabolism of PC.

Choline kinase, responsible for the phosphorylation of choline to phosphocholine as the first step of the CDP-choline pathway for the biosynthesis of phosphatidylcholine, has been recognized as a new target for anticancer therapy. Crystal structures of human choline kinase in its apo, ADP-and phosphocholine-bound complexes, respectively, reveal the molecular details of the substrate binding sites. ATP binds in a cavity where residues from both the N-and C-terminal lobes contribute to form a cleft, while the choline-binding site constitutes a deep hydrophobic groove in the C-terminal domain with a rim composed of negatively charged residues. Upon binding of choline, the enzyme undergoes conformational changes independently affecting the N-terminal domain and the ATP-binding loop. From this structural analysis and comparison with other kinases, and from mutagenesis data on the homologous C. elegans choline kinase, a model of the ternary ADP•Phosphocholine complex was built that reveals the molecular basis for the phosphoryl transfer activity of this enzyme.

A novel family of compounds derivative of 1,1′-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene)) bis(methylene))-bispyridinium or-bisquinolinium bromide (10a-l) containing a pair of oxygen atoms in the spacer of the linker between the biscationic moieties, were synthesized and evaluated as inhibitors of choline kinase against a panel of cancer-cell lines. The most promising compounds in this series were 1,1′-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))bis(4-(dimethylamino)pyridinium) bromide (10a) and 1,1′-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))-bis(7-chloro-4-(pyrrolidin-1-yl)quinolinium) bromide (10l), which inhibit human choline kinase (ChoKα1) with IC 50 of 1.0 and 0.92 μM, respectively, in a range similar to that of the previously reported biscationic compounds MN58b and RSM932A. Our compounds show greater antiproliferative activities than do the reference compounds, with unprecedented values of GI 50 in the nanomolar range for several of the cancer-cell lines assayed, and more importantly they present low toxicity in non-tumoral cell lines, suggesting a cancer-cell-selective antiproliferative activity. Docking studies predict that the compounds interact with the choline-binding site in agreement with the binding mode of most previously reported biscationic compounds. Moreover, the crystal structure of ChoKα1 with compound 10a reveals that this compound binds to the choline-binding site and mimics HC-3 binding mode as never before. Cancer is a worldwide health threat and the second leading cause of mortality in developed countries 1,2. Since many of the current treatments still prove toxic and/or lead to drug resistance, there is a strong demand for the discovery and development of effective new cancer therapies 3. Protein kinases have emerged as one of the most important types of targets in cancer-drug discovery due to their major roles in regulating cell growth and survival and many other cell functions 4,5. An abnormal kinase signaling network underlies the development and progression of tumors, and thus the targeted inhibition of protein kinases has become an attractive strategy in cancer treatment (for a recent review see Gross et al. 6) and in the last decade the intense development in the field has led to different kinase inhibitors that have been approved for use in clinical therapy.

Choline kinase-α expression and activity are increased in multiple human neoplasms as a result of growth factor stimulation and activation of cancer-related signaling pathways. The product of choline kinase-α, phosphocholine, serves as an essential metabolic reservoir for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the production of lipid second messengers. Using in silico screening for small molecules that may interact with the choline kinase-α substrate binding domain, we identified a novel competitive inhibitor, N-(3,5-dimethylphenyl)-2-[[5-(4-ethylphenyl)-1H-1,2,4-triazol-3-yl]sulfanyl] acetamide (termed CK37) that inhibited purified recombinant human choline kinase-α activity, reduced the steady-state concentration of phosphocholine in transformed cells, and selectively suppressed the growth of neoplastic cells relative to normal epithelial cells. Choline kinase-α activity is required for the downstream production of phosphatidic acid, a promoter of several Ras signaling pathways. CK37 suppressed MAPK and PI3K/AKT signaling, disrupted actin cytoskeletal organization, and reduced plasma membrane ruffling. Finally, administration of CK37 significantly decreased tumor growth in a lung tumor xenograft mouse model, suppressed tumor phosphocholine, and diminished activating phosphorylations of ERK and AKT in vivo. Together, these results further validate choline kinase-α as a molecular target for the development of agents that interrupt Ras signaling pathways, and indicate that receptor-based computational screening should facilitate the identification of new classes of choline kinase-α inhibitors. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite&#39;s growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidyl...

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase β is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase α is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb −/− mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A 2 in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb −/− mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb −/− mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb −/− mice is due to abundant choline kinase α and the stable homeostasis of phosphatidylcholine.

Objectives: Polymorphisms in the TCRA and P2RY11, two immune related genes, are associated with narcolepsy in Caucasians and Asians. In contrast, CPT1B/CHKB polymorphisms have only been shown to be associated with narcolepsy in Japanese, with replication in a small group of Koreans. Our aim was to study whether these polymorphisms are associated with narcolepsy and its clinical characteristics in Chinese patients with narcolepsy. Methods: We collected clinical data on 510 Chinese patients presenting with narcolepsy/hypocretin deficiency. Patients were included either when hypocretin deficiency was documented (CSF hypocretin-1 6 110 pg/ml, n = 91) or on the basis of the presence of clear cataplexy and HLA-DQB1⁄0602 positivity (n = 419). Genetic data was compared to typing obtained in 452 controls matched for geographic origin within China. Clinical evaluations included demographics, the Stanford Sleep Inventory (presence and age of onset of each symptom), and Multiple Sleep Latency Test (MSLT) data. Results: Chinese narcolepsy was strongly and dose dependently associated with TCRA (rs1154155C) and P2RY11 (rs2305795A) but not CPT1B/CHKB (rs5770917C) polymorphisms. CPT1B/CHKB polymorphisms were not associated with any specific clinical characteristics. TCRA rs1154155A homozygotes (58 subjects) had a later disease onset, but this was not significant when corrected for multiple comparisons, thus replication is needed. CPT1B/CHKB or P2RY11 polymorphisms were not associated with any specific clinical characteristics. Conclusions: The study extends on the observation of a strong multiethnic association of polymorphisms in the TCRA and P2RY11 with narcolepsy, but does not confirm the association of CPT1B/CHKB (rs5770917) in the Chinese population.

The genomes of Treponema denticola and Treponema pallidum contain a gene, licCA, which is predicted to encode a fusion protein containing choline kinase and CTP:phosphocholine cytidylyltransferase activities. Because both organisms have been reported to contain phosphatidylcholine, this raises the possibility that they use a CDP-choline pathway for the biosynthesis of phosphatidylcholine. This report shows that phosphatidylcholine is a major phospholipid in T. denticola, accounting for 35-40% of total phospholipid. This organism readily incorporated [14C]choline into phosphatidylcholine, indicating the presence of a choline-dependent biosynthetic pathway. The licCA gene was cloned, and recombinant LicCA had choline kinase and CTP:phosphocholine cytidylyltransferase activity. The licCA gene was disrupted in T. denticola by erythromycin cassette mutagenesis, resulting in a viable mutant. This disruption completely blocked incorporation of either [14C]choline or 32Pi into phosphatidylc...

The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, although the molecular mechanisms are not resolved. To address this, we performed a functional siRNA of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes that both positively and negatively regulate AT1R-EGFR transactivation. Many candidates comprised components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, had not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but not following direct stimulation of the EGFR with EGF, indicating that these genes function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R-EGFR transactivation. CHKA also ...

Some enzymes responsible for de novo synthesis of phosphatidylcholine and the reacylation of phosphatidylcholine to disaturated phosphatidylcholine have been examined in isolated rat type II alveolar cells. Compared to the enzyme levels found in whole lung homogenate, levels of choline kinase, choline phosphotransferase and lysolecithin acyltransferase are enriched 10 fold in isolated type II alveolar cells. No enrichment of cholinephosphate cytidyltransferase was observed suggesting that this enzyme may be rate limiting in phosphatidylcholine biosynthesis.

Mouse peritoneal macrophages turn over their membrane components rapidly. The biosynthetic pathway for phosphatidylcholine synthesis from choline can be inhibited in a non-toxic manner by a series of diphenylsulfones. The parent compound of this series, dapsone, and its derivative, I-[4-(4.sulfanilyl)phenyl lurea (AUS), inhibit the synthesis of lecithin from choline by mouse peritoneal macrophages in a dose-and time-dependent manner. This inhibition of choline incorporation does not appear to be due to an effect on transport of choline into the cells nor is it an effect on general membrane synthesis. In addition, the sulfones inhibit the incorporation of [ "P Iphosphate into phospatidylcholine but do not significantly inhibit its incorporation into sphingomyelin. phosphatidylinositol or phosphatidylethanolamine. This inhibition of lecithin synthesis is reversible and appears to require functionally intact membranes.

A series of bispyridinium compounds were synthesized by a short sequence of reactions from symmetric diamides. All compounds were tested for their antiproliferative activity against HT-29, a cell line derived from a human colon adenocarcinoma, and their inhibitory activity against choline kinase (ChoK), a novel anticancer molecular target already in clinical trials. Most of the compounds analyzed showed good antiproliferative activities, in the micromolar range, with the identification of promising lead molecules as a new family of potential inhibitors of ChoK.

The choline-containing teichoic and lipoteichoic acids play an important part in cell wall metabolism of Streptococcus pneumoniue. We propose that a choline kinase enzyme has a role in the synthesis of these antigens. The presence of this enzyme was demonstrated in cell free extracts of S. pneumoniue by measuring the fall in ATP concentration due to phosphorylation of choline. Genomic DNA of S. pneumoniae hybridised with a probe consisting of an internal fragment of the choline kinase gene of Saccharomyces cererlisiae and one consisting of the choline binding domain of 1ytA.

The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKa and ChoKb isoforms, the first one with two different variants of splicing. Recently ChoKa has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKb in carcinogenesis has been reported. Methodology/Principal Findings: Here we compare the in vitro and in vivo properties of ChoKa1 and ChoKb in lipid metabolism, and their potential role in carcinogenesis. Both ChoKa1 and ChoKb showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKb display an ethanolamine kinase role, ChoKa1 present a dual choline/ ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKa1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKb overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKa1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKb mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKa1 than ChoKb. This study represents the first evidence of the distinct metabolic role of ChoKa and ChoKb isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition.

In Arabidopsis (Arabidopsis thaliana), the At1g74310 locus encodes for caseinolytic protease B-cytoplasmic (ClpB-C)/heat shock protein100 protein (AtClpB-C), which is critical for the acquisition of thermotolerance, and At1g74320 encodes for choline kinase (AtCK2) that catalyzes the first reaction in the Kennedy pathway for phosphatidylcholine biosynthesis. Previous work has established that the knockout mutants of these genes display heat-sensitive phenotypes. While analyzing the AtClpB-C promoter and upstream genomic regions in this study, we noted that AtClpB-C and AtCK2 genes are head-to-head oriented on chromosome 1 of the Arabidopsis genome. Expression analysis showed that transcripts of these genes are rapidly induced in response to heat stress treatment. In stably transformed Arabidopsis plants harboring this intergenic sequence between head-to-head oriented green fluorescent protein and β-glucuronidase reporter genes, both transcripts and proteins of the two reporters were ...

Exposure of mammalian cells to UV irradiation stimulates phosphatidylcholine hydrolysis and activates the transcription factor AP-1. Since phosphocholine (PCho), a phospholipid metabolite, is a potential regulator of mitogenesis and carcinogenesis, we examined the ...

The effect of intake of ethanol on choline-and ethanolamine kinase activities in liver and brain tissues of mice was studied over a period of 31 days. With a single dose of ethanol there was an initial slight increase in hepatic kinase activity, while brain kinase activity decreased. The activities returned to the normal level after 24 h. Intragastric intubation of 1 ml of 30% aqueous ethanol daily for 1 month to mice led to a decrease in activity of both enzymes in both tissues.

Small interfering RNA (siRNA) is routinely used as a biological tool to silence specific genes, and is under active investigation in cancer treatment strategies. Noninvasive magnetic resonance spectroscopy (MRS) provides the ability to assess the functional effects of siRNA-mediated gene silencing in cultured cancer cells, and following nanoparticle-based delivery in tumors in vivo. Here we describe the use of siRNA to downregulate choline kinase, a critical enzyme in choline phospholipid metabolism of cancer cells and tumors, and the use of 1 H MRS of cells and 1 H magnetic resonance spectroscopic imaging (MRSI) of tumors to assess the efficacy of the downregulation.

Choline, a component of the wall teichoic acid of Streptococcus pneumoniae, was converted to cytidine diphosphocholine via choline phosphate by enzymes which were identified in cell-free extracts of the pneumococcus. The first enzyme, choline kinase, was investigated in some detail. It appeared to have a pH optimum of 7.3 to 7.4 and was stimulated by Mg2+. Kinetic studies gave an apparent Michaelis constant (Km) for ATP of I mM, and for choline of 0.19 mM, with Vmax values of 3 nmol min-1 (mg protein)-1 and 0.5 nmol min-1 (mg protein)-1 respectively. The second enzyme, CDPcholine pyrophosphorylase was specific for CTP and had a requirement for Mg2+ with an optimum at 7 mM.

lterations in cell metabolism represent a common cause of human diseases [1]. Changes in lipid profiles are not an exception to this rule, including those lipids that are produced via the choline kinase (ChoK) pathway. ChoK catalyzes the conversion of choline to phosphocholine...