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At least 73 records · Page 4Linked to original sources
PubMed2006

Insight into the inhibition of human choline kinase: homology modeling and molecular dynamics simulations.

A homology model of human choline kinase (CK-alpha) based on the X-ray crystallographic structure of C. elegans choline kinase (CKA-2) is presented. Molecular dynamics simulations performed on CK-alpha confirm the quality of the model, and also support the putative ATP and choline binding sites. A good correlation between the MD results and reported CKA-2 mutagenesis assays has been found for the main residues involved in catalytic activity. Preliminary docking studies performed on the CK-alpha model indicate that inhibitors can bind to the binding sites of both substrates (ATP and choline). A possible reason for inhibition of choline kinase by Ca(2+) ion is also proposed.

Amino Acid Sequence↗
PubMed2002

Regulation of choline kinase activity by Ras proteins involves Ral-GDS and PI3K.

Ras proteins are molecular switches that control signaling pathways critical in the onset of a variety of human cancers. The signaling pathways activated by Ras proteins are those controlled by its direct effectors such as the serine-threonine protein kinase Raf-1, the exchange factor for other GTPases Ral-GDS, and the lipid kinase PI3K. As a consequence of Ras activation, a number of additional enzymes are affected, including several members of the serine-threonine intracellular proteins kinases as well as enzymes related to phospholipid metabolism regulation such as phospholipases A2 and D, and choline kinase. The precise mechanisms by which ras oncogenes impinge into these later molecules and their relevance to the onset of the carcinogenic process is still not fully understood. Here we have investigated the mechanism of regulation of choline kinase by Ras proteins and found no direct link between PLD and choline kinase activation. We provide evidence that Ras proteins regulate the activity of choline kinase through its direct effectors Ral-GDS and PI3K, while the Raf pathways seems to be not relevant in this process. The importance of Ras-dependent activation of choline kinase is discussed.

3T3 Cells↗
PubMed1979

Choline kinase and ethanolamine kinase activity in the cytosol of nerve endings from rat forebrain.

Both choline kinase and ethanolamine kinase are present in the cytosol of nerve endings prepared from rat brain are the products of their action, phosphocholine (84 nmol/g fresh wt. of brain) and phosphoethanolamine (190 nmol/g fresh wt. of brain). In contrast with the enzymes from the cytosol of whole brain, both are as equally active at pH 7.5 as 9.0. Determination of kinase activity in membrane-containing tissue samples at pH9 gives low values because of the activity of alkaline phosphatase. Choline kinase, but not ethanolamine kinase, requires Mg2+ in excess of that required for the formation of the MgATP complex and is inhibited by an excess of free ATP. The Km for choline is 2.6mM and for ethanolamine is 2.2mM. The differing requirements for ATP and Mg2+ and the inhibition of choline kinase, but not ethanolamine kinase, by hemicholinium-3 suggest either the presence of two separate enzymes or two different active sites on the same enzyme.

Animals↗
PubMed1999

Increased choline kinase activity and elevated phosphocholine levels in human colon cancer.

Nuclear magnetic resonance spectroscopy has detected elevated phosphocholine levels in human tumor tissues and cells, and in cells that were transformed with the activated Ha-ras gene and stimulated in vitro with growth-promoting factors such as platelet-derived growth factor, epidermal growth factor, and phorbol ester. However, the mechanism of the elevation and the function of the increased phosphocholine levels have not been clearly demonstrated. We studied phosphocholine levels enzymatically and analyzed the activity of choline kinase, which catalyzes the phosphorylation of choline to produce phosphocholine, in human colon cancer and adenoma. Both choline kinase activity and phosphocholine levels were increased in colon cancer and adenoma tissue. The activation of choline kinase and the increased levels of choline kinase alpha were partly responsible for the elevated phosphocholine levels. This study suggests that choline kinase might play a role in growth promotion or signal transduction in carcinogenesis.

Adult↗
PubMed1983

Actinomycin D-sensitive induction of choline kinase by carbon tetrachloride intoxication in rat liver.

A single intraperitoneal dose(1 ml/kg body weight) of carbon tetrachloride (CCl4) caused a rapid and drastic induction of choline kinase activity in rat liver cytosol. The administration of either cycloheximide or actinomycin D completely blocked the CCl4-mediated induction of choline kinase activity, indicating that the elevated activity could be due to the change in the enzyme level. The pretreatment of rats with phenobarbital did not cause any significant effect on hepatic choline kinase induction by CCl4, suggesting that the induction may not be directly related to the metabolic rate of CCl4. A considerable part of induced form(s) of choline kinase appeared not to be a form present in the liver of untreated rats. The contribution of adrenals to the CCl4-mediated hepatic choline kinase induction could be ruled out.

Adrenalectomy↗
PubMed1978

Control of phosphatidylcholine synthesis and the regulatory role of choline kinase in rat liver. Evidence from essential-fatty acid-deficient rats.

Choline kinase and phosphocholine cytidylytransferase catalyse the rate-limiting steps of the cytidine pathway for the synthesis of phosphatidylcholine [Infante (1977) Biochem. J. 167, 847--849]. Essential-fatty acid deficiency induces a 3.5-fold increase in the specific activity of choline kinase, whereas the specific activity of the cytidylytransferase remains unchanged in rat liver. This change in specific activity accounts for the calculated increase in flux through the cytidine pathway produced in vivo by the same dietary state [Trewhella & Collins (1973 Biochim. Biophys. Acta 296, 34--50], thus confirming the fact that choline kinase has a regulatory role in the cytidine pathway for the synthesis of phosphatidylcholine.

Animals↗
PubMed1992

Cloning of a human choline kinase cDNA by complementation of the yeast cki mutation.

A human choline kinase cDNA was cloned by complementation of the yeast choline kinase mutation, cki, from a human glioblastoma cDNA expression library. The deduced sequence of the human enzyme comprised 456 amino acids with a calculated relative molecular mass of 52,065. The human enzyme resembled the rat liver enzyme over the entire sequence. It also resembled the yeast enzyme in the carboxy-terminal region, but not much in the amino-terminal region.

Amino Acid Sequence↗
PubMed1985

Complete co-purification of choline kinase and ethanolamine kinase from rat kidney and immunological evidence for both kinase activities residing on the same enzyme protein(s) in rat tissues.

Choline kinase and ethanolamine kinase were completely co-purified from rat kidney cytosol through acid treatment, ammonium sulfate fractionation, DEAE-cellulose column chromatography, Sephadex G-150 gel filtration followed by choline-Sepharose affinity chromatography. The final preparation appeared to be highly homogeneous with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ishidate, K., Nakagomi, K. and Nakazawa, Y. (1984) J. Biol. Chem. 259, 14706-14710). Throughout the purification steps, the ratio of ethanolamine kinase activity to choline kinase activity was almost constant in a range of 0.3-0.4, which strongly indicated that, in rat kidney, both activities could reside on a single enzyme protein. The rabbit polyclonal antibody raised against highly purified rat kidney choline (ethanolamine) kinase protein inhibited both choline and ethanolamine kinase activities in a parallel manner in crude enzyme preparations not only from rat kidney, but also from rat liver, lung and intestinal cytosols. The results, together with our previous findings, suggested strongly that, in rat tissues, at least large portions of both kinase activities are present on the same enzyme protein(s). The kinetic properties of both kinase reactions with the highly purified kidney enzyme were compared in some detail and the overall result suggested that choline kinase and ethanolamine kinase activities may not have a common active site on a single enzyme protein.

Adenosine Triphosphate↗
PubMed1995

Early increase in choline kinase activity upon induction of the H-ras oncogene in mouse fibroblast cell lines.

The effects of expression of the H-ras oncogene on phosphatidylcholine metabolism were examined in C3H10T1/2 and NIH3T3 cells expressing ras constitutively or under the control of inducible promoters. Cell lines expressing ras under the control of the mouse metallothionein promoter and the Escherichia coli lac operator/repressor system and an NIH3T3 cell line stably transfected with the ras oncogene were studied. Phosphocholine levels were elevated in the cells constitutively expressing ras and were increased 4-6 h upon induction in the inducible cell lines. Glycerophosphocholine, which is elevated five- to sixfold in constitutively transfected ras cells, did not increase at early times of induction, suggesting the absence of increased phosphatidylcholine degradation via a phospholipase A. Choline kinase activity increased within 4-6 h upon induction and correlated well with the increase in phosphocholine levels. This increase in phosphocholine levels could be prevented by the addition of hemicholinium-3, a competitive inhibitor of choline kinase. Expression of activated c-raf or v-raf also increased choline kinase activity, suggesting that the induction of choline kinase by ras is downstream of the ras/raf interaction. Long-term and short-term labeling experiments failed to detect evidence for increased phospholipase C activity. These results suggest that the increase in choline kinase activity observed in cells expressing ras is an early, integral part of ras transformation and is the main contributor to increased phosphocholine levels accompanying morphological changes.

3T3 Cells↗
PubMed1982

Noncorrelation of choline kinase or ATP citrate lyase with cholinergic activity in rat brain.

The activity of choline acetyltransferase was used as an index of cholinergic structures in regions of rat brain. The activities of ATP citrate lyase and choline kinase correlated poorly with cholinergic activity in whole tissue fractions, contrasting with the good correlation between acetylcholinesterase and choline acetyltransferase. Choline acetyltransferase was preferentially localised in synaptosomes prepared from regions of high (striatum) or intermediate (cortex, medulla oblongata/pons) cholinergic activity. In general, this was not true for either choline kinase or ATP citrate lyase.

ATP Citrate (pro-S)-Lyase↗
PubMed1982

Diethylstilbestrol treatment increases the amount of choline kinase in rooster liver.

Studies have been performed on the mechanism by which diethylstilbestrol stimulates the activity of choline kinase in livers from cockerels. The enzyme was purified 700-900-fold by affinity chromatography. The increased enzyme activity could not be accounted for by diethylstilbestrol alteration of the kinetic constants of the enzyme. Rabbit antibody was raised to the purified enzyme. Titration studies with antiserum demonstrated a 2-fold increase in the amount of choline kinase in diethylstilbestrol-treated cytosol, which correlated with a 2-fold elevation of the activity of the enzyme. We conclude that diethylstilbestrol stimulates the activities of choline kinase in cockerel liver by corresponding increase in the amount of enzyme.

Animals↗
PubMed1994

N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulphonamide (H-89) inhibits incorporation of choline into phosphatidylcholine via inhibition of choline kinase and has no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase.

We have shown previously that N-[2-bromocinnamyl(amino)-ethyl]-5-isoquinolinesulphonamide (H-89), a selective inhibitor of cyclic-AMP-dependent protein kinase (PKA), inhibits phosphatidylcholine biosynthesis in HeLa cells. In the present study, we elucidated the mechanism underlying the described inhibition. Treatment of cells with 10 microM H-89 had no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase. However, H-89 slightly affected the distribution of cytidylyltransferase between cytosol and membranes, but the cellular 1,2-diacylglycerol content was not influenced. Furthermore, pulse-chase experiments revealed that H-89 did not affect cytidylyltransferase activity. Instead, H-89 inhibited choline kinase, the enzyme catalysing the first step in the CDP-choline pathway. In the presence of 10 microM H-89, choline kinase activity was inhibited by 36 +/- 7.6% in vitro. Additionally, the phosphorylation of choline to phosphocholine was inhibited by 30 +/- 3% in cell-culture experiments. This inhibitory effect could be partly prevented by simultaneous addition of 10 microM forskolin, indicating that choline kinase is regulated in part by PKA activity.

Cell Membrane↗
PubMed1981

Increased choline kinase activity in the rat superior cervical ganglion after axonal injury.

The activity of choline kinase (CK) was examined in the rat superior cervical ganglion (SCG) during development and following postganglionic axotomy. The highest specific enzyme activity (nmol phosphorylcholine/mg protein/h) 52 +/- 8, is observed 5 d before birth, then it rapidly decreases by about 50%, reaching at the day of birth levels observed in the ganglion throughout life. During development the total enzyme activity per ganglion is increased steadily until it reaches a 5-fold increase which parallels the increase in protein content. Following axotomy the enzyme activity per ganglion is rapidly increased by about 2-fold between 1 and 5 d postoperative and then gradually decreases reaching control levels at 30 d. The transient increase in enzyme activity parallels the increase in protein content of the axotomized ganglia. The peak increase in enzyme activity coincides with the peak chromatolytic response of the axotomized ganglion. We conclude that choline kinase activity is transiently increased within neurons after axonal injury, and that this event represents an effort of the nerve cell body to enhance its phosphatidylcholine biosynthesis essential for new membrane synthesis during the regeneration of the cut axon.

Aging↗
PubMed1966

Choline kinase and phosphorylcholine phosphatase in plants.

Choline kinase was present in barley and wheat roots and leaves of barley, wheat, tobacco, spinach and squash plants. The kinase was purified 25-fold from spinach leaves. The enzyme had a broad pH optimum between 7.5 and 10.0. Mg(++) was required for activity and in the presence of Mg(++) the enzyme was relatively stable. Maximum enzyme activity was obtained when the Mg(++): ATP ratio was 1:1. The K(m) was 1 x 10(-4)m. The kinase from leaves was similar to that from rapeseed or from yeast, except that the leaf and seed enzymes were not inhibited by compounds which attach sulfhydryl groups. Only a very slow hydrolysis of phosphorylcholine by similar plant extracts was observed. This phosphatase activity was purified 200- or 300-fold and appeared to be caused by a nonspecific acid phosphatase. The activity of both the kinase and the phosphatase did not seem sufficient to account for the rapid equilibration of the large phosphorylcholine reservoir of plants with exogenous P(32)-labeled orthophosphate.

In Vitro Techniques↗
PubMed1998

Molecular cloning of mouse choline kinase and choline/ethanolamine kinase: their sequence comparison to the respective rat homologs.

Complementary DNAs homologous to a rat 42-kDa choline/ethanolamine kinase [C. Aoyama et al., Biochim. Biophys. Acta 1390 (1998) 1-7] and to a 50-kDa choline kinase [T. Uchida and S. Yamashita, J. Biol. Chem. 267 (1992) 10156-10162] were isolated from a 17-day post coitum mouse embryo cDNA library and their sequences were compared with the two murine species, respectively. The nucleotide sequence homology (within the coding sequence) between mouse and rat 50-kDa choline kinases (96.0%) was considerably higher than that between their 42-kDa choline/ethanolamine kinases (92.4%). Northern blot and RT-PCR studies on several rat tissues demonstrated that both isozymes are expressed ubiquitously with the highest level in testis.

Amino Acid Sequence↗
PubMed2000

Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

3T3 Cells↗
PubMed2006

Generation and characterization of monoclonal antibodies against choline kinase alpha and their potential use as diagnostic tools in cancer.

Choline kinase alpha (ChoKalpha) is a metabolic enzyme involved in the synthesis of phosphatidylcholine, recently implicated in cancer onset since it is overexpressed in a variety of human cancers such as mammary, lung, colorectal and prostate adenocarcinomas. Furthermore, overexpression of ChoKalpha in human HEK293T cells confers them oncogenic properties with the induction of tumors after subcutaneous injection in nude mice. ChoKalpha levels in tumor samples have been analyzed using polyclonal antibodies and Western blotting. These techniques have considerable limitations and do not allow for a precise and efficient evaluation of the real significance of ChoK overexpression in human carcinogenesis. We developed a set of monoclonal antibodies with high specificity and sensitivity against ChoKalpha, and characterized their properties. We provide evidence that the newly generated MoAbs against ChoKalpha have potential use in cancer diagnosis by conventional immunohistochemistry techniques.

Animals↗