Choline kinase

Choline kinase (also known as CK, ChoK and choline phosphokinase) is an enzyme which catalyzes the first reaction in the choline pathway for phosphatidylcholine (PC) biosynthesis. This reaction involves the transfer of a phosphate group from adenosine triphosphate (ATP) to choline in order to form phosphocholine.

ATP + choline ADP + O-phosphocholine
Choline kinase
Identifiers
EC no.2.7.1.32
CAS no.9026-67-9
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Thus, the two substrates of this enzyme are ATP and choline, whereas its two products are adenosine diphosphate (ADP) and O-phosphocholine. Choline kinase requires magnesium ions (+2) as a cofactor for this reaction.[1] This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. The first detailed investigation of the enzyme was conducted by McCamen in 1962, where it was shown that the brain is the richest source of the enzyme in mammalian tissue. A related enzyme, ethanolamine kinase, tends to co-purify with choline kinase leading to a suggestion that the two activities are mediated by two distinct active sites on a single protein.[2] The systematic name of this enzyme class is ATP:choline phosphotransferase. These enzymes participate in glycine, serine and threonine metabolism and glycerophospholipid metabolism. In mammalian cells, the enzyme exists as three isoforms: CKα-1, CKα-2 and CKβ. These isoforms are encoded by two separate genes, CHKA and CHKB and are only active in their homodimeric, heterodimeric and oligomeric forms.[3]

Structural studies

As of late 2007, six structures have been solved for this class of enzymes, with PDB accession codes 1NW1, 2CKO, 2CKP, 2CKQ, 2I7Q, and 2IG7.

CKα-2 originating from C. elegans, is a dimeric enzyme with each monomer being composed of two domains. The active site is located between the two domains (see figure below). Its overall structure is similar to members of the eukaryotic protein kinase family. Mammalian choline kinases exists in either dimeric or tetrameric forms in solution.[4][5] Structural studies carried out on CKα-2 have implied that the conserved residues in the CK family of enzymes could possibly play a vital role in substrate binding as well as in the stabilization of catalytically important residues.[6]

An enlarged view of the residues involved in the dimer interface between the S-shaped loop of the yellow subunit and the loop following helix A and strand 4 of the cyan subunit. Only residues that are involved in direct salt bridges, hydrogen bonds, or van der Waals interactions are shown. Salt bridges and hydrogen bonds, dashed lines; labels of residues from the yellow subunit, red; labels of residues from the cyan subunit, blue.[6]

Mechanism

Although not much is known about the mechanism by which choline kinase reacts, the recent advancement in the elucidation of the structure of the enzyme has provided scientists with much more insight than they had previously. Since the structure of CK is very similar to that of the eukaryotic protein kinase family, the location of ATP and choline binding pockets have been proposed. These are shown in the figures below.

Proposed ATP binding site

In this figure, there is a similarity between APH(3′)-IIIa, an aminoglycoside phosphotransferase and CK.

Proposed choline binding site

Propositions for this mechanism have been made based on mechanistic studies done on eukaryotic protein kinases. It has been proposed that in the CKα-2 mechanism, ATP binds first, followed by choline, and then the transfer of the phosphoryl group takes place. The product O-phosphocholine is then released, followed by the release of ADP.[7]

Evolution

After closely studying the structurally similar enzymes, CKα-2, APH(3′)-IIIa, and PKA, researchers observed that PKA had less insertions to its structural core compared to the other enzymes. Against this background, it is believed that CKα-2 have evolved from PKA to have more structural elements attached to it.[8]

Biological function

Choline kinase catalyzes the formation of phosphocholine, the committed step in phosphatidylcholine biosynthesis. Phosphatidylcholine is the major phospholipid in eukaryotic membranes. Phosphatidylcholine is important for a variety of function in eukaryotes such as facilitating the transport of cholesterol through the organism, acting as a substrate for the production of second messengers and as a cofactor for the activity of several membrane-related enzymes.[9] CK also plays a vital role in the production of sphingomyelin, another important membrane phospholipid and in the regulation of cell growth.[10]

The production of phosphocholine from CK is necessary for the signal transduction pathways related to mitogenesis. It has also been found that CK plays a critical role in the proliferation of human mammary epithelial cells.[11]

Choline kinase α as protein chaperone

Choline kinase α can act as a protein chaperone.[12] Kinase can function as chaperone and there may be other kinases that may function as chaperone that are yet to be identified. Choline kinase α (CKα) is overexpressed in prostate cancer where it physically interacts with the androgen receptor (AR), a major driver of prostate cancer. By disabling the function of CHKA researchers were able to inhibit AR function and prostate cancer growth.

In vivo studies carried out using CKα-1 and CKβ isoforms suggest that each isoform might be involved in different biochemical pathways. CKβ plays a major role in the catalysis of the phosphorylation of ethanolamine while CKα-1 catalyzes the phosphorylation of both choline and ethanolamine.[13] ShRNA mediated in vivo depletion of CKα has been shown to decrease the growth of prostate tumor xenografts[12]

Disease relevance

Oncogenic activity and CKα-1

Overexpression of CKα-1 has been found to be associated with cancer. Recent studies carried out on cancer cell lines have shown that CKα-1 is over-expressed in breast cancer cells. This leads to an accumulation of phosphocholine in the breast and causes malignancy.[14]

Studies using colon, human lung and prostate carcinomas also revealed that CK is upregulated by overexpression of CKα-1 in these cells compared to the normal, non-cancerous cells.[15]

One possible explanation for this is that CKα-1 aids in the regulation of protein kinase B phosphorylation, particularly at the Serine-473 end. Consequently, high levels of expression and activity of CKα-1 promotes cell growth and survival.[16] Based on the observation that increased activity of CKα-1 is related to cancer, CKα-1 has promising use as a tumor biomarker and in diagnosing and following the progression of tumors. All human cancer cells have shown increased levels of this particular enzyme.[15]

Muscular dystrophy and CKβ

It has been shown, using CKβ knockout mice models, that a defect in the CKβ activity leads to a decrease in the phosphatidylcholine (PC) content in the hindlimb muscle. This, however, does not affect the phosphoethanolamine (PE) content.[17]

The net effect is then that the PC/PE ratio decreases and this leads to impaired membrane integrity in the liver.[18] This compromised membrane potential leads to malfunctioning of the mitochondria. Although CK is required for the biosynthesis of PC, CK is normally present in excess and so is not generally considered the rate-limiting step.[19] Researchers have concluded, however, that due to the reduced activity of CK seen in the hindlimb muscle of the CKβ knockout mice model, CK is probably the rate-limiting enzyme in skeletal muscles. This suggests that defect in CKβ may lead to a decrease in PC synthesis in the muscles resulting in muscular dystrophy.[17] These results suggest that CK could possibly play a vital role in the homeostasis of PC.[20]

References
  1. Wu G, Vance DE (August 2010). "Choline kinase and its function". Biochemistry and Cell Biology. 88 (4): 559–64. doi:10.1139/O09-160. PMID 20651826.
  2. Spanner S, Ansell GB (1978). "Choline and Ethanolamine Kinase Activity in the Cytoplasm of Nerve Endings from Rat Forebrain". Enzymes of Lipid Metabolism. Advances in Experimental Medicine and Biology. Vol. 101. pp. 237–45. doi:10.1007/978-1-4615-9071-2_23. ISBN 978-1-4615-9073-6. PMID 208357.
  3. Aoyama C, Liao H, Ishidate K (May 2004). "Structure and function of choline kinase isoforms in mammalian cells". Progress in Lipid Research. 43 (3): 266–81. doi:10.1016/j.plipres.2003.12.001. PMID 15003397.
  4. Porter TJ, Kent C (January 1990). "Purification and characterization of choline/ethanolamine kinase from rat liver". The Journal of Biological Chemistry. 265 (1): 414–22. doi:10.1016/S0021-9258(19)40246-9. PMID 2152925.
  5. Uchida T, Yamashita S (May 1992). "Molecular cloning, characterization, and expression in Escherichia coli of a cDNA encoding mammalian choline kinase". The Journal of Biological Chemistry. 267 (14): 10156–62. doi:10.1016/S0021-9258(19)50213-7. PMID 1577786.
  6. Peisach D, Gee P, Kent C, Xu Z (June 2003). "The crystal structure of choline kinase reveals a eukaryotic protein kinase fold". Structure. 11 (6): 703–13. doi:10.1016/S0969-2126(03)00094-7. PMID 12791258.
  7. McKay GA, Wright GD (October 1995). "Kinetic mechanism of aminoglycoside phosphotransferase type IIIa. Evidence for a Theorell-Chance mechanism". The Journal of Biological Chemistry. 270 (42): 24686–92. doi:10.1074/jbc.270.42.24686. PMID 7559583.
  8. Hon WC, McKay GA, Thompson PR, Sweet RM, Yang DS, Wright GD, Berghuis AM (June 1997). "Structure of an enzyme required for aminoglycoside antibiotic resistance reveals homology to eukaryotic protein kinases". Cell. 89 (6): 887–95. doi:10.1016/S0092-8674(00)80274-3. PMID 9200607. S2CID 13251696.
  9. Kent C (1990). "Regulation of phosphatidylcholine biosynthesis". Progress in Lipid Research. 29 (2): 87–105. doi:10.1016/0163-7827(90)90010-I. PMID 1965552.
  10. Janardhan S, Srivani P, Sastry GN (2006). "Choline kinase: an important target for cancer". Current Medicinal Chemistry. 13 (10): 1169–86. doi:10.2174/092986706776360923. PMID 16719778.
  11. Ramírez de Molina A, Báñez-Coronel M, Gutiérrez R, Rodríguez-González A, Olmeda D, Megías D, Lacal JC (September 2004). "Choline kinase activation is a critical requirement for the proliferation of primary human mammary epithelial cells and breast tumor progression". Cancer Research. 64 (18): 6732–9. doi:10.1158/0008-5472.CAN-04-0489. PMID 15374991. S2CID 14747787.
  12. Asim M, Massie CE, Orafidiya F, Pértega-Gomes N, Warren AY, Esmaeili M, et al. (May 2016). "Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target". Journal of the National Cancer Institute. 108 (5): djv371. doi:10.1093/jnci/djv371. PMC 4849803. PMID 26657335.
  13. Gallego-Ortega D, Gómez del Pulgar T, Valdés-Mora F, Cebrián A, Lacal JC (2011). "Involvement of human choline kinase alpha and beta in carcinogenesis: a different role in lipid metabolism and biological functions". Advances in Enzyme Regulation. 51 (1): 183–94. doi:10.1016/j.advenzreg.2010.09.010. PMID 21035492..
  14. Eliyahu G, Kreizman T, Degani H (April 2007). "Phosphocholine as a biomarker of breast cancer: molecular and biochemical studies". International Journal of Cancer. 120 (8): 1721–30. doi:10.1002/ijc.22293. PMID 17236204. S2CID 24802164.
  15. Ramírez de Molina A, Rodríguez-González A, Gutiérrez R, Martínez-Piñeiro L, Sánchez J, Bonilla F, Rosell R, Lacal J (August 2002). "Overexpression of choline kinase is a frequent feature in human tumor-derived cell lines and in lung, prostate, and colorectal human cancers". Biochemical and Biophysical Research Communications. 296 (3): 580–3. doi:10.1016/S0006-291X(02)00920-8. PMID 12176020.
  16. Chua BT, Gallego-Ortega D, Ramirez de Molina A, Ullrich A, Lacal JC, Downward J (December 2009). "Regulation of Akt(ser473) phosphorylation by choline kinase in breast carcinoma cells". Molecular Cancer. 8: 131. doi:10.1186/1476-4598-8-131. PMC 2806310. PMID 20042122.
  17. Wu G, Sher RB, Cox GA, Vance DE (May 2009). "Understanding the muscular dystrophy caused by deletion of choline kinase beta in mice". Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1791 (5): 347–56. doi:10.1016/j.bbalip.2009.02.006. PMID 19236939.
  18. Li Z, Agellon LB, Allen TM, Umeda M, Jewell L, Mason A, Vance DE (May 2006). "The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis". Cell Metabolism. 3 (5): 321–31. doi:10.1016/j.cmet.2006.03.007. PMID 16679290.
  19. Vance, Dennis; Vance, Jean (2008). "Phospholipid biosynthesis in eukaryotes". In Vance, Dennis E.; Vance, Jean E. (eds.). Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier. pp. 213–244. doi:10.1016/B978-044453219-0.50010-6. ISBN 978-0-444-53219-0.
  20. Mori N, Glunde K, Takagi T, Raman V, Bhujwalla ZM (December 2007). "Choline kinase down-regulation increases the effect of 5-fluorouracil in breast cancer cells". Cancer Research. 67 (23): 11284–90. doi:10.1158/0008-5472.CAN-07-2728. PMID 18056454.

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