Characterization of glycerophosphodiesterase 4-interacting molecules Gαq/11 and Gß, which mediate cellular lysophospholipase D activity.

We previously purified lysophospholipase D (lysoPLD), which hydrolyzes lysophosphatidylcholine (lysoPC) to lysophosphatidic acid (LPA), from rat brain and identified the heterotrimeric G protein subunits Gαq and Gß1 in the lysoPLD active fractions. Tag-affinity purified Gαq exhibits lysoPLD activity but a mutant that affected cellular localization or interaction with the Gß subunit reduced lysoPLD activity. Size exclusion chromatography revealed that active lysoPLD is a much higher molecular mass complex than is heterotrimeric G protein, suggesting the presence of other components. Liquid chromatography-tandem mass spectrometry of lysoPLD purified from rat brain identified glycerophosphodiesterase 4 (GDE4), recently reported as lysoPLD, in the same fraction as G proteins. The overexpressed and tag-purified Gαq fractions, which exhibit lysoPLD activity, contained GDE4. Exogenously expressed GDE4 was co-immunoprecipitated with endogenous Gαq and Gß and exhibited high lysoPLD activity. The results of confocal microscopy and cell fractionation experiments indicated that exogenously expressed GDE4 in cells mainly localized at the endoplasmic reticulum and partially co-localized with Gαq protein at the plasma membrane. Proteinase K protection assay results suggested that the catalytic domain of GDE4 faces the lumen/extracellular space. Mutations at the conserved amino acids in the C-terminus cytoplasmic regions amongst GDE1, 4 and 7, dramatically suppressed GDE4 enzyme activities. When both the Gαq and Gα11 genes in Neuro2A cells were disrupted using the CRISPR-Cas9 system, endogenous lysoPLD activity was partially reduced but rescued by overexpression of Gαq. These results suggest that GDE4 is a new effector of G protein signaling that produces bioactive phospholipid LPA and/or modulates membrane homeostasis.

StarD7 Protein Deficiency Adversely Affects the Phosphatidylcholine Composition, Respiratory Activity, and Cristae Structure of Mitochondria.

Phosphatidylcholine (PC) is a major phospholipid of mitochondria, comprising 40-50% of both the outer and the inner membranes. However, PC must be imported from its production organelles because mitochondria lack the enzymes essential for PC biosynthesis. In a previous study, we found that StarD7 mediates the intracellular transfer of PC to mitochondria. Therefore, in this study, we analyzed the contribution of StarD7 to the maintenance of mitochondrial phospholipid content and function using siRNA-mediated knockdown and knock-out (KO) of the StarD7 gene in HEPA-1 cells. Real time analysis of respiratory activity demonstrated that the oxygen consumption rate and activity of mitochondrial complexes were impaired in StarD7-KD cells. To confirm these results, we established StarD7-KO HEPA-1 cells by double nicking using CRISPR/Cas9n. As expected, StarD7-KD and -KO cells showed a significant reduction in mitochondrial PC content. The ATP level and growth rate of KO cells were notably lower compared with wild-type cells when cultured in glucose-free galactose-containing medium to force cells to rely on mitochondrial ATP production. In KO cells, the level of the MTCO1 protein, a primary subunit of complex IV, was reduced without a concomitant decrease in its mRNA, but the level was restored when StarD7-I was overexpressed. StarD7-KO cells showed impaired formation of the mitochondrial supercomplexes and exhibited a disorganized cristae structure, with no changes in optic atrophy 1 protein. These findings indicate that StarD7 plays important roles in maintaining the proper composition of mitochondrial phospholipids as well as mitochondrial function and morphogenesis.

Transcriptional suppression of CTP:phosphoethanolamine cytidylyltransferase by 25-hydroxycholesterol is mediated by nuclear factor-Y and Yin Yang 1.

Pcyt2 (CTP:phosphoethanolamine cytidylyltransferase) is the rate-limiting enzyme in mammalian PE (phosphatidylethanolamine) biosynthesis. Previously, we reported that Pcyt2 mRNA levels increased in several types of cells after serum starvation, an effect that could be suppressed by supplementation with low-density lipoprotein or 25-HC (25-hydroxycholesterol). Transcription of Hmgcr, which encodes 3-hydroxy-3-methylglutaryl-CoA reductase, is also suppressed by 25-HC in the same dose-dependent manner. Nevertheless, a sterol-regulatory element was not detected in the Pcyt2 promoter region. The important element for transcriptional control of Pcyt2 by 25-HC (1.25 µM) was determined to reside between -56 and -36 on the basis of analysis with several Pcyt2 promoter deletion-luciferase reporters in NIH 3T3 cells. Using the yeast one-hybrid system, we found that NF-Y (nuclear factor-Y) binds at C(-37)CAAT(-41) and YY1 (Yin Yang1) binds at C(-42)AT(-40) in the Pcyt2 promoter. Endogenous NF-Y and YY1 bind clearly and competitively to these sites and are important for basal Pcyt2 transcription. Moreover, NF-Y binds to the Hmgcr promoter at C(-14)CA(-12) in gel-shift analysis, and suppression of the basal luciferase activity of the Hmgcr promoter-reporter construct (-30/+61) by 25-HC was abolished when C(-14)CA(-12) was mutated. Furthermore, transcriptional suppression of Pcyt2 by 25-HC was reduced following knockdown targeting of NF-YA or YY1. ChIP analysis revealed that 25-HC inhibited the interaction between NF-Y and RNA polymerase II on the Pcyt2 and Hmgcr promoters. On the basis of these results, we conclude that NF-Y and YY1 are important for the basal transcription of Pcyt2 and that NF-Y is involved in the inhibitory effects of 25-HC on Pcyt2 transcription.

Choline kinase alpha expression during RA-induced neuronal differentiation: role of C/EBPß.

Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-ß (C/EBPß) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPß expression. Elevated levels of C/EBPß bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation.

A congenital muscular dystrophy with mitochondrial structural abnormalities caused by defective de novo phosphatidylcholine biosynthesis.

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.

The heterotrimeric G protein subunits Gα(q) and Gß(1) have lysophospholipase D activity.

In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gα(q) and Gß(1) respectively. When FLAG-tagged Gα(q) or Gß(1) was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg(2+) dependency and substrate specificity of Gα(q) were similar to those of lysoPLD purified from the rat brain. Mutation of Gα(q) at amino acids Lys(52), Thr(186) or Asp(205), residues that are predicted to interact with nucleotide phosphates or catalytic Mg(2+), dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gα(q) overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated K(m) and V(max) values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 µM and 0.16 µmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gα(q) and Gß(1) as an enzyme with lysoPLD activity. Tag-purified Gα(11) also exhibited a high lysoPLD activity, but Gα(i) and Gα(s) did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gα(q) and Gα(11) siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies.

Identification of nuclear localization and nuclear export signals in Ets2, and the transcriptional regulation of Ets2 and CTP:phosphocholine cytidylyltransferase alpha in tetradecanoyl-13-acetate or macrophage-colony stimulating factor stimulated RAW264 cells.

PC is made via the CDP-choline pathway, in which CTP:phosphocholine cytidylyltransferase alpha (CTalpha), encoded by Pcyt1a, is the rate-limiting enzyme whose mRNA expression is strictly regulated. Previously, we reported that Ets1 enhanced and Net repressed CTalpha transcription by binding at the Ets binding site (-49/-47) in the Pcyt1a promoter. In this study, we asked if an Ets1 analogue, Ets2, also regulates CTalpha transcription and investigated the importance of its nuclear localization signal (NLS) and nuclear export signal (NES). Ets2 is primarily detected in the nucleus. Various mutated Ets2 proteins fused with enhanced green fluorescent protein were constructed to identify the NLS and NES in Ets2. Mutation of Ets2 at amino acids 404-410 results in a protein that is evenly distributed in the cell. Interestingly, an Ets2 protein deleted at the C-terminus (amino acids 1-392 present) was localized to the cytoplasm and site-specific mutation in the region 364-372 of this construct resulted in cytoplasmic and nuclear distribution. These results suggest that the NLS in Ets2 is between amino acids 404 and 410, and that the NES is between amino acids 364 and 372. Ets2 enhanced, but the mutant forms of Ets2 had little effects on the transcription of a CTalpha-reporter construct. When RAW264 cells, murine macrophage cell-line, were stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) or macrophage-colony stimulating factor, the transcription of CTalpha was enhanced accompanied by increased mRNA of Ets2. These results suggest that the induction of Ets2 is important for CTalpha transcription by TPA and macrophage-colony stimulating factor.

The phosphatidylcholine transfer protein StarD7 is important for myogenic differentiation in mouse myoblast C2C12 cells and human primary skeletal myoblasts.

StarD7 is a phosphatidylcholine (PC)-specific lipid transfer protein essential for the maintenance of mitochondrial PC composition, morphogenesis, and respiration. Here, we studied the role of StarD7 in skeletal myoblast differentiation using mouse myoblast C2C12 cells and human primary myoblasts. Immunofluorescence and immuno-electron microscopy revealed that StarD7 was distributed in the cytosol, inner mitochondria space, and outer leaflet of the outer mitochondrial membrane in C2C12 cells. Unlike human kidney embryonic cell line HEK293 cells, the mitochondrial proteinase PARL was not involved in the processing and maturation of StarD7 in C2C12 cells. StarD7 was constantly expressed during myogenic differentiation of C2C12 cells. The siRNA-mediated knockdown of StarD7 in C2C12 cells and human primary myoblasts significantly impaired myogenic differentiation and reduced the expression of myomaker, myomerger and PGC-1α. The reduction in mitochondrial PC levels and oxygen consumption rates, decreased expression of myomaker, myomerger and PGC-1α, as well as impaired myogenic differentiation, were completely restored when the protein was reintroduced into StarD7-knockout C2C12 cells. These results suggest that StarD7 is important for skeletal myogenesis in mammals.

Side-chain oxysterols suppress the transcription of CTP: Phosphoethanolamine cytidylyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase by inhibiting the interaction of p300 and NF-Y, and H3K27 acetylation.

CTP: phosphoethanolamine cytidylyltransferase (Pcyt2) is the rate-limiting enzyme in mammalian phosphatidylethanolamine (PE) biosynthesis. Previously, we reported that increasedPcyt2 mRNA levels after serum starvation are suppressed by 25-hydroxycholesterol (HC) (25-HC), and that nuclear factor-Y (NF-Y) is involved in the inhibitory effects. Transcription of Hmgcr, which encodes 3-hydroxy-3-methylglutaryl-CoA reductase, is suppressed in the same manner. However, no typical sterol regulatory element (SRE) was detected in the Pcyt2 promoter. We were therefore interested in the effect of 25-HC on the modification of histones and thus treated cells with histone acetyltransferase inhibitor (anacardic acid) or histone deacetylase inhibitor (trichostatin A). The suppressive effect of 25-HC on Pcyt2 and Hmgcr mRNA transcription was ameliorated by trichostatin A. Anacardic acid, 25-HC and 24(S)-HC suppressed their transcription by inhibiting H3K27 acetylation in their promoters as evaluated by chromatin immunoprecipitation (ChIP) assays. 27-HC, 22(S)-HC and 22(R)-HC also suppressed their transcription, but 7α-HC, 7ß-HC, the synthetic LXR agonist T0901317 and cholesterol did not. Furthermore, 25-HC inhibited p300 recruitment to the Pcyt2 and Hmgcr promoters, and suppressed H3K27 acetylation. 25-HC in the medium was easily conducted into cells. Based on these results, we concluded that 25-HC (and other side-chain oxysterols) in the medium was easily transferred into cells, suppressed H3K27 acetylation via p300 recruitment on the NF-Y complex in the Pcyt2 and Hmgcr promoters, and then suppressed transcription of these genes although LXR is not involved.

Induction of choline kinase alpha by carbon tetrachloride (CCl4) occurs via increased binding of c-jun to an AP-1 element.

The mechanism by which treatment of mice with CCl4 induces an increase in choline kinase alpha has been investigated. Nuclear run on assays demonstrated a major increase in the transcript for choline kinase alpha in livers from mice 3 h and 6 h after administration of CCl4 compared to vehicle (olive oil). 5'deletion analyses of choline kinase alpha promoter-luciferase constructs expressed in Hepa-1 cells identified a promoter element between -875 and -866 that was nearly identical to an AP-1 consensus site. Mutation of this AP-1 site caused a striking decrease in the expression of choline kinase alpha promoter-luciferase constructs. Electromobility shift assays with nuclear extracts from mouse liver demonstrated that c-Jun, but not c-fos, bound oligonucleotides with the AP-1 site. The amount of c-jun bound was greatly increased when hepatic nuclear extracts from mice treated with CCl4 were used. Chromatin immunoprecipitation assays confirmed that c-jun binds to the choline kinase alpha promoter. The results from these studies provide strong evidence that the choline kinase alpha promoter has a distal element (-875/-867) that binds c-jun and the binding of c-jun is enhanced by treatment with CCl4.

Deletion and alanine mutation analyses for the formation of active homo- or hetero-dimer complexes of mouse choline kinase-alpha and -beta.

Choline kinase (CK) is the first-step regulatory enzyme for the biosynthesis of phosphatidylcholine in all mammalian cells. It exists as at least three isoforms (alpha1, alpha2 and beta) that are encoded by two separate genes termed ck-alpha and ck-beta. The active enzyme has been proposed to consist of either their homo- or hetero-dimeric forms. Here, we report on the identification of several essential domains and amino acid residues involved in their active dimer formation. Full-length cDNAs or their truncated or alanine-mutated versions for mouse CK-alpha1 and CK-beta tagged with either HA or Myc at their N-termini were expressed in COS-7 cells. Each dimer formation was analyzed by immuno-precipitation followed by Western blotting. Kinetic analysis for CK reaction was performed with different expression products. Both the N-terminal domain-1 and C-terminal portions (E424-K430 for CK-alpha1 and Q379-K385 for CK-beta) were shown to be critical for the formation of active homo- or hetero-dimer complex. Interestingly, D320 in the CK-motif of CK-alpha1 was found to be essential for alpha1/alpha1 homo-dimerization but not for alpha1/beta hetero-dimerization. A mutation of the corresponding D276 of CK-beta to A276 did not show any effect on either its homo- or hetero-dimerization but it caused a strong inhibition of CK activity in either case.

Purification and characterization of lysophospholipase D from rat brain.

A lysophospholipase D (lysoPLD) was purified to apparent homogeneity from rat brain nuclear fractions using 1-[(14)C]palmitoyl-glycerophosphorylcholine as a substrate. The abundance of autotaxin (ATX), a secretory lysoPLD, was also estimated for each fraction. The nuclear fraction had relatively high levels of lysoPLD activity but weak immunoreactivity with an anti-ATX antibody. LysoPLD activity was further purified 5550-fold by sequential chromatography. The final preparation migrated as a single band with a molecular weight of 35,000. Anti-ATX antibodies did not cross-react with the purified enzyme. Moreover, enzyme activity was highest at pH 7.0-7.5 and requires Mg(2+). The Km and Vmax values for 1-palmitoyl-glycerophosphorylcholine were 176 microM and 0.3 micromol/min/mg, respectively. The purified enzyme hydrolyzed saturated forms of LPC more robustly than unsaturated forms. The enzyme could hydrolyze platelet-activating factor (PAF) to the same extent as 16:0-LPC, and showed a higher activity toward lysoPAF (1-O-hexadecyl-2-lyso-glycerophosphorylcholine). These results suggested that the lysoPLD purified from rat brain nuclear fractions in this work is a novel enzyme that hydrolyzes lysoPAF, PAF, and LPC to liberate choline.

Structure and function of choline kinase isoforms in mammalian cells.

Choline kinase (CK) catalyzes the first phosphorylation reaction in the CDP-choline pathway for the biosynthesis of phosphatidylcholine (PC), yielding phosphocholine (P-Cho) from choline and ATP in the presence of Mg(2+). This enzyme exists in mammalian cells as at least three isoforms that are encoded by two separate genes termed ck-alpha and ck-beta. Each isoform is not active in its monomeric form. The active enzyme consists of either their homo- or hetero-dimeric (or oligomeric) forms. In recent years, the roles of CK in cell growth and cell stress/defense mechanisms have been intensely investigated. These functions of CK do not seem to be directly related to the net PC biosynthesis but predict another important role of this enzyme in certain cell physiology. This review summarizes briefly the recent progress of mammalian CK study which will include the gene structure of each isoform and its possible transcriptional regulation, the active configuration of the enzyme, induction of the particular isoform in chemically induced cell stress, and the possible role of this enzyme as well as of its reaction product, P-Cho, in cell growth and other cellular physiology.

Muscular dystrophy with large mitochondria associated with mutations in the CHKB gene in three British patients: extending the clinical and pathological phenotype.

Three patients with CHKB deficient muscular dystrophy are described which broadens the previously described phenotype. Blood smear in one patient showed Jordans anomaly (vacuolated leukocytes). Gastrointestinal features occurred in two patients and there appeared to be acute deterioration with infection/general anaesthesia. Brain imaging showed no structural changes but brain magnetic resonance proton spectroscopy (MRS) demonstrated significant reduction in choline:N-acetyl aspartate and choline:creatine ratios in keeping with a general decrease in the amount of choline and phosphocholine-based substrate. Muscle pathology showed either myopathic or dystrophic features, uneven oxidative enzyme staining, COX deficient fibres and peripherally located large mitochondria. CHKB activity was reduced in all three patients and complex 1 activity was significantly reduced in one patient.

Early embryonic lethality caused by disruption of the gene for choline kinase alpha, the first enzyme in phosphatidylcholine biosynthesis.

Choline kinase alpha (CK-alpha) is one of two mammalian enzymes that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid, phosphatidylcholine. We created mice lacking CK-alpha with an embryonic stem cell line containing an insertional mutation in the gene for CK-alpha (Chka). Embryos homozygous for the mutant Chka allele were recovered at the blastocyst stage, but not at embryonic day 7.5, indicating that CK-alpha is crucial for the early development of mouse embryos. Heterozygous mutant mice (Chka(+/-)) appeared entirely normal in their embryonic development and gross anatomy, and they were fertile. Although choline kinase activity was decreased by approximately 30%, the amount of phosphatidylcholine in cells and the levels of other enzymes involved in phosphatidylcholine biosynthesis were unaffected. Phosphatidylcholine biosynthesis measured by choline incorporation into hepatocytes was also not compromised in Chka(+/-) mice. Enhanced levels of choline and attenuated levels of phosphocholine were observed in both the livers and testes of Chka(+/-) mice. Triacylglycerol and cholesterol ester were elevated approximately 2-fold in the livers, whereas neutral lipid profiles in plasma were similar in Chka(+/-) and wild-type (Chka(+/+)) mice. Thus, Chka is an essential gene for early embryonic development, but adult mice do not require full expression of the gene for normal levels of phosphatidylcholine.

A rostrocaudal muscular dystrophy caused by a defect in choline kinase beta, the first enzyme in phosphatidylcholine biosynthesis.

Muscular dystrophies include a diverse group of genetically heterogeneous disorders that together affect 1 in 2000 births worldwide. The diseases are characterized by progressive muscle weakness and wasting that lead to severe disability and often premature death. Rostrocaudal muscular dystrophy (rmd) is a new recessive mouse mutation that causes a rapidly progressive muscular dystrophy and a neonatal forelimb bone deformity. The rmd mutation is a 1.6-kb intragenic deletion within the choline kinase beta (Chkb) gene, resulting in a complete loss of CHKB protein and enzymatic activity. CHKB is one of two mammalian choline kinase (CHK) enzymes (alpha and beta) that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid phosphatidylcholine. While mutant rmd mice show a dramatic decrease of CHK activity in all tissues, the dystrophy is only evident in skeletal muscle tissues in an unusual rostral-to-caudal gradient. Minor membrane disruption similar to dysferlinopathies suggest that membrane fusion defects may underlie this dystrophy, because severe membrane disruptions are not evident as determined by creatine kinase levels, Evans Blue infiltration, and unaltered levels of proteins in the dystrophin-glycoprotein complex. The rmd mutant mouse offers the first demonstration of a defect in a phospholipid biosynthetic enzyme causing muscular dystrophy, representing a unique model for understanding mechanisms of muscle degeneration.

Expression and characterization of the active molecular forms of choline/ethanolamine kinase-alpha and -beta in mouse tissues, including carbon tetrachloride-induced liver.

Choline/ethanolamine kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.