Choline kinase, phosphorylation

Choline kinase phosphorylates choline to give a phosphocholine and participates in glycine, serine and threonine metabolism and glycerophospholipid metabolism. Hemicholinium-7 is the prototypical tool compound used to inhibit CHK. Based on inhibitor studies, it has been proposed that CHK is important for the regulation of cell proliferation. Inhibition of choline kinase is also used to target plasmodium and develop novel antimalarials. A series of papers on pyridinium based inhibitors have been published, but no disclosures of more drug-like molecules have been made. 

The majority of PC is synthesized in mammalian cells by the CDP-choline or Kennedy pathway in the endoplasmic reticulum (Eigiue 1). In this pathway, choline taken up from the external medium or released in the cytosol by breakdown of choline containing compoimds, is first converted to phosphocholine by the enzyme choline kinase (CK) (Ishidate, 1997). There are two isoforms of CK cloned which both can convert also ethanolamine to phosphoethanolamine, albeit with a lesser affinity (Aoyama et al 2000). Alternatively phosphocholine can be generated by enzymes that preferentially phosphorylate ethanolamine and are therefore designated ethanolamine kinases (EK). As yet also two different EKs are known (EKI 1 and 2 Lykidis etal., 2001). 

The enzymatic radioassay method for the analysis of acetylcholine and choline in brain tissue has been reported by Reid et al. [210]. The method describes the determination of nanogram amounts of acetylcholine and choline in as little as 10 mg of brain tissue, involves isolation of acetylcholine by high-voltage paper electrophoresis, alkaline hydrolysis of acetylcholine to choline, and conversion of this into [32P]-phosphoryl choline in the presence of choline kinase and [y32P] ATP. The labeled derivative is isolated by column chromatography on Bio-Rad AG1-X8 resin, using Tris buffer solution as the eluent. Cerenkov radiation from 32P is counted (at 33% efficiency) in a liquid scintillation spectrometer. The amount of phosphorylcholine is proportional to the amount of choline over the range of 0.08-8.25 nmol. 

Phosphatidylcholine is degraded by phospholipases that cleave preferentially at specific bonds (Chapter 18). Choline released is phosphorylated by choline kinase and reutilized in phosphatidylcholine synthesis. However, in liver mitochondria, choline is also oxidized to betaine (N-trimethylglycine) ... 

Once choline has entered the cell, its normal fate is rapid phosphorylation by choline kinase (Fig. 3). In neurons choline is also converted to the neurotransmitter, acetylcholine. Choline can also be oxidized to betaine [-00C-CH2-N (CH3)3] in the liver and kidney. In liver, betaine is an important donor of methyl groups for methionine biosynthesis. Betaine is produced in mitochondria into which choline is transported by a specific transporter on the inner membranes. Next, choline is oxidized to betaine aldehyde by choline dehydrogenase on the inner leaflet of the mitochondrial inner membranes and the subsequent conversion to betaine is catalyzed by betaine-aldehyde dehydrogenase in the mitochondrial matrix. Betaine can be transported into kidney medulla by a betaine transporter. In renal medulla and many plants and organisms, betaine accumulates as an osmolyte (a small organic solute that accumulates in response to hypertonicity without adverse effects to the cell or organism) (J.S. Handler, 1992). Hypertonicity of the renal medulla is important for the kidney s ability to concentrate urine. 

The conversion of choline to choline-P was first demonstrated in yeast extracts by Wittenberg and Komberg (more famous for his contributions to DNA replication) in 1953. The enzyme was purified (K. Ishidate, 1984) from rat kidney and shown also to phosphorylate ethanolamine [5]. This kinase is now referred to as choline kinase p. Three isoforms of choline kinase have been identified (al oi2, and P). Northern analyses indicate that the mRNA encoding choline kinase al is most abundant in testis. Choline kinase o2 is a splice variant of choline kinase al. The choline kinase a and P genes (Chka and Chkb, respectively) have been characterized. The length of Chka is 40 kb whereas Chkb is only... 

The CT reaction usually limits the rate of PC biosynthesis. The first evidence in support of this conclusion was drawn from the relative pool sizes of the aqueous precursors (in rat liver, choline = 0.23 mM, phosphocholine =1.3 mM, CDP-choline = 0.03 mM). Calculation of these values assumes that 1 g wet tissue is equivalent to 1 ml and that there is no compartmentation of the pools. The second assumption may not be valid as there is evidence for compartmentation of PC precursors (M.W. Spence, 1989). The concentration of phosphocholine is 40-fold higher than that of CDP-choline, consistent with a bottleneck in the pathway at the reaction catalyzed by CT. Pulse-chase experiments illustrate this bottleneck more vividly. After a 0.5 h pulse of hepatocytes with [methyl- H]choline, more than 95% of radioactivity in the precursors of PC was in phosphocholine, with the remainder in choline and CDP-choline. When the radioactivity was chased with unlabeled choline, labeled phosphocholine was quantitatively converted to PC (Fig. 5). The radioactivity in CDP-choline remained low during the chase and CDP-choline was rapidly converted to PC. There was minimal radioactivity in choline which suggests that choline is immediately phosphorylated after it enters the cell. It is important to note that if a cell or tissue is in a steady state, pool sizes and reaction rates do not change. Thus, although the rate of PC synthesis is determined by the CT reaction, the rates of the reactions catalyzed by choline kinase and cholinephosphotransferase are the same as that of the reaction catalyzed by CT, otherwise, the pool sizes of precursors would change. For example, if the choline kinase reaction were faster than the CT reaction, the amount of phosphocholine would increase. Thus, CT sets the pace of the pathway. 

Fig. 9 NMR spectral changes revealed by a 5 mm solution of hyperpolarized choline upon undergoing phosphorylation by 0.5 units of choline kinase, (a) Emergence of the new phosphocholine resonance shown by directly detected single-pulse N NMR spectroscopy experiments, (b) Emergence of the H NMR resonance associated with the methylenes in the C2-position of phosphocholine, (c) Comparison between the expected enzyme kinetics of kinase with results afforded by the N- ( ) and H-detected ( ) hyperpolarized experiments, as derived from the relative peak ratios of the NMR peaks in (a) and (b). The straight line illustrates the best fit of the combined set of data points, and corresponds to an initial phosphorylation rate of 0.3 mM min under these conditions. Reproduced with permission from [55]...

As shown in Figs 11.13 and 11.14, phos-phatidycholine (the major plant and animal glycerophospholipid) and phosphatidylethanolamine are both synthesized by a CDP-base pathway. This is the major route of their formation in both plants (Harwood, 1979 Moore, 1982) and animals (Bell and Coleman, 1980 Spanner, 1982). Three enzyme steps are required. Firstly, the base is phosphorylated by a kinase enzyme. Choline kinase (EC 2.7.1.32) and ethanolamine kinase (EC 2.7.1.82) are soluble enzymes and have been purified from several tissues. The activities may reside in the same protein (Ulane et al.y 1977) although separate enzymes have been purified from rat liver (Brophy and Vance, 1976) and soybean seeds (Wharfe and Harwood, 1979). 

The pH profile and requirement for Mg-ATP were similar to those for choline kinase however, activities could be clearly resolved by electrophoresis which revealed a single peak of ethanolamine kinase and two peaks of choline kinase activity. Phosphorylation of ethanolamine was less in the presence of monomethyl- and dimethylethanolamine, but it was not clear whether these compounds were phosphorylated by the.enzyme. Choline had no effect on the phosphorylation of ethanolamine. Waring and Laties (1977) supplied dimethylethanolamine to aging slices of potato tuber and found marked synthesis of phosphatidyldimethylethanolamine (PDME), while the accumulation of PC and PE was inhibited. It appeared that dimethylethanolamine was phosphorylated and metabolized as an analogue of choline and/or ethanolamine. 

Choline, an amino alcohol, is the precursor of phospholipids, which are ubiquitous components of cell membranes. Choline can be labeled with "C (50) or F (51,52) and is taken up by tumor cells via an active transport mechanism. It is subsequently trapped through phosphorylation by choline kinase to... 

Synthesis of PE and PC from preexisting choline and ethanolamine These synthetic pathways involve the phosphorylation of choline or ethanolamine by kinases, followed by conversion to the activated form, CDP-choline or CDP-ethanolamine. Finally, choline-phosphate or ethanolamine-phosphate is transferred from the nucleotide (leaving CMP) to a molecule of diacylglycerol (see Figure 17.5). 

The second phase of phospholipid synthesis in eukaryotes. Choline or ethanolamine enters the cell via active transport mechanisms and is immediately phosphorylated by the enzyme, choline (ethanolamine) kinase. The phosphorylated derivatives of choline and ethanolamine... 

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