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J Biol Chem, Vol. 275, Issue 2, 1377-1383, January 14, 2000

Pantothenate Kinase Regulation of the Intracellular Concentration of Coenzyme A^*

Charles O. Rock^§, Robert B. Calder, Mohammad A. Karim, and Suzanne Jackowski^§¶

From the  Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 and the ^§ Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway in bacteria and is thought to play a similar role in mammalian cells. We examined this hypothesis by identifying and characterizing two murine cDNAs that encoded PanK. The two cDNAs were predicted to arise from alternate splicing of the same gene to yield different mRNAs that encode two isoforms (mPanK1 and mPanK1) with distinct amino termini. The predicted protein sequence of mPanK1 was not related to bacterial PanK but exhibited significant similarity to Aspergillus nidulans PanK. mPanK1 was most highly expressed in heart and kidney, whereas mPanK1 mRNA was detected primarily in liver and kidney. Pantothenate was the most abundant pathway component (42.8%) in normal cells providing clear evidence that pantothenate phosphorylation was a rate-controlling step in CoA biosynthesis. Enhanced mPanK1 expression eliminated the intracellular pantothenate pool and triggered a 13-fold increase in intracellular CoA content. mPanK1 activity in vitro was stimulated by CoA and strongly inhibited by acetyl-CoA illustrating that differential modulation of mPanK1 activity by pathway end products also contributed to the management of CoA levels. These data support the concept that the expression and/or activity of PanK is a determining factor in the physiological regulation of the intracellular CoA concentration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pantothenate kinase (PanK)¹ (ATP:D-pantothenate 4'-phosphotransferase, EC 2.7.1.33) catalyzes the first committed step in the universal biosynthetic pathway leading to CoA (for review see Ref. 1). Phosphopantothenate is rapidly metabolized to CoA, which participates as an acyl group carrier in the tricarboxylic acid cycle, fatty acid metabolism, and numerous other reactions of intermediary metabolism (2). The 4'-phosphopantetheine portion of CoA is an essential prosthetic group in a number of enzyme systems including the acyl carrier protein components, bacterial and eukaryotic fatty acid synthases (3), citrate lyase (4), ferrichrome synthetase from Apsergillus quadricinctus (5), and malonate decarboxylase of Malonomonas rubra (6).

PanK is the rate-controlling enzyme in CoA biosynthesis in Escherichia coli (1). E. coli is capable of de novo pantothenate biosynthesis, and additionally a sodium-dependent permease actively transports exogenous pantothenate into the cell (7-9). Metabolic labeling experiments established that the utilization, rather than the supply of pantothenate, controls the level of CoA (10). E. coli mutants with temperature-sensitive PanK activity are also temperature-sensitive for CoA biosynthesis and growth (11). The PanK gene of E. coli (coaA) was cloned by functional complementation of this mutant and was identical to a previously sequenced allele called rts (12-14). E. coli PanK (bPanK) is a homodimer of 36-kDa subunits that exhibit highly positive cooperative ATP binding and mediate sequential ordered catalysis with ATP as the leading substrate (15). CoA and its thioesters inhibit bPanK activity by competitive binding to the ATP site (15, 16). Nonesterified CoA is the most potent inhibitor of bPanK in vitro and in vivo, whereas acetyl-CoA is about 20% as effective as CoA (16). This feedback regulation is a primary mechanism by which bacteria control the cellular CoA level.

PanK is also proposed to be the master regulator of CoA biosynthesis in mammalian cells (for review see Ref. 17). Metabolic labeling experiments in the rat heart model support a role for PanK in controlling flux through the CoA biosynthetic pathway (18, 19). The metabolic state of the animal (starvation or feeding) (20-24), pathological states (diabetes) (25, 26), and drugs (clofibrate) (24, 27, 28) all significantly alter the cellular levels of CoA, and these fluctuations are reflected by concomitant changes in the level of tissue PanK activity (29, 30). Since CoA and its thioesters are important regulators of several key enzymes in intermediary metabolism, such as pyruvate dehydrogenase, adjustments in the level of CoA and acetyl-CoA that occur in the examples cited above contribute to the metabolic alterations observed during fasting, in diabetes, and following treatment with hypolipidemic drugs. CoA and CoA thioesters also inhibit crude preparations of mammalian (18, 31-36) and plant (37) PanK enzymes. In contrast to bacteria, acetyl-CoA is significantly more effective than CoA at inhibiting mammalian PanK in cell extracts and partially purified preparations. A recent detailed study with a purified eukaryotic PanK from A. nidulans (aPanK) found that acetyl-CoA was a potent aPanK inhibitor, whereas CoA was ineffective (38). The bacterial PanK and aPanK do not exhibit a significant degree of similarity in their protein sequences reflecting the sharp contrast in the specificity of feedback regulation between the two proteins. The goal of the present study was to identify and characterize a mammalian PanK and test whether the expression level of this enzyme influences the regulation of intracellular CoA content, and to determine if CoA or acetyl-CoA is the most important feedback regulator of mammalian PanK activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Sources of supplies were as follows: American Radiolabeled Chemicals, D-[1-¹⁴C]pantothenate (specific activity, 53.5 mCi/mmol); Bio-Rad, Bradford dye-binding protein assay solution; Promega, Klenow fragment; Analtech Inc., 250-µm Silica Gel H thin layer chromatography plates; Qiagen, Qiaquick kit; NEN Life Science Products, [-³²P]dCTP (specific activity, 3000 Ci/mmol); Fisher, Scintisafe 30%; Life Technologies, Inc., LipofectAMINE^TM reagent; Promega, restriction endonucleases and T4 DNA ligase; Qiagen, P100 columns; Sigma, CoA, acetyl-CoA, and malonyl-CoA; and Whatman, DE81 filter circles. All other materials were reagent grade or better.

Cloning the mPanK1 cDNAs-- The A. nidulans PanK sequence was used to search the EST data base for related clones. This yielded clone AA030321 expressed in fetal tissue that contains the common region of mPanK1. mPanK1- (AA014914 expressed in mouse placenta) and mPanK1-specific (AI181055 expressed in mouse liver and AA105778 expressed in mouse kidney) EST clones were subsequently identified using AA030321.

3'-Rapid amplification of cDNA ends was performed to determine the 3'-untranslated region of the mPanK1 cDNA using reagents supplied by Life Techologies, Inc. Poly(A)⁺ RNAs (0.2 µg) from mouse brain, heart, kidney, liver, or skeletal muscle tissues (CLONTECH) were used as template for the first-strand cDNA synthesis together with an oligo(dT)-containing adapter primer 5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3' and Superscript TMII reverse transcriptase. The first-strand cDNA was used as a template for the PCR using the mPanK1-specific primer 5'-GATCTAGTGATCTTTTGGATCAAGG-3' and the abridged universal amplification primer 5'-GGCCACGCGTCGACTAGTAC-3'. PCR products of the same size were amplified from all five tissues. The products were subcloned into the TA vector pCR2.1 (Invitrogen) and sequenced on both strands using M13 forward and reverse primers. The cDNA sequences of the 3'-untranslated regions were identical in all of the clones.

The mPanK1 expression vector (pmPanK1) was constructed by digesting EST clone AA105778 with BstXI. This product was first blunted by digestion with mung bean nuclease and the product was then digested with NheI. This fragment containing the entire mPanK1 open reading frame was cloned into plasmid pcDNA3.1 that had been digested with EcoRV and NheI. The clone was verified by DNA sequencing.

DNA Sequencing-- The mPanK cDNA sequences were determined on both strands by automated DNA sequencing using ABI 373A automated fluorescent sequencing equipment at the Molecular Resource Center of St. Jude Children's Research Hospital.

Pantothenate Kinase Assays-- Enzyme preparation and assays were performed as described previously (16). The pantothenate kinase-specific activities in cell lysates were calculated as a function of protein concentration. Assays were linear with respect to both time and protein input. Protein concentrations were measured by the method of Bradford (39) with bovine -globulin as a standard. Standard assays contained D-[1-¹⁴C]pantothenate (45.5 µM; specific activity 55 mCi/mmol), ATP (2.5 mM, pH 7.0), MgCl₂ (2.5 mM), Tris-HCl (0.1 M, pH 7.5), and 15 µg of protein from a soluble cell extract (see below) in a total volume of 40 µl. The mixture was incubated for 10 min at 37 °C, and the reaction was stopped by depositing a 30-µl aliquot onto a Whatman DE81 ion-exchange filter disc that was washed in three changes of 1% acetic acid in 95% ethanol (25 ml/disc) to remove unreacted pantothenate. 4'-Phosphopantothenate was quantitated by counting the dried disc in 3 ml of scintillation solution.

Transfection Experiments-- COS-7 cells were grown in 100-mm dishes to 80% confluency in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% glutamine. Transfections using LipofectAMINE^TM reagent were performed according to the manufacturer's instructions. Briefly, 10 µg of plasmid and 60 µl of LipofectAMINE^TM reagent were diluted separately into 0.8 ml of serum-free medium. The two solutions were combined and incubated at room temperature for 45 min. Next, 6.4 ml of serum-free medium was added to each tube, and the diluted solution was overlaid onto cells that had been previously rinsed with serum-free medium. The cells and reagents were incubated at 37 °C for 5 h and then 8 ml of growth medium containing twice the normal amount of serum was added. The medium was replaced 24 h after the start of the transfection procedure.

At 24 h post-transfection, cells were labeled at 37 °C for 24 h with D-[1-¹⁴C]pantothenate (2 µM, specific activity 55 mCi/mmol) in pantothenate-free Dulbecco's modified Eagle's medium and dialyzed serum. Cells were washed with phosphate-buffered saline, harvested by scraping and centrifugation, and stored as pellets at 20 °C. Pantothenate metabolites were separated on Silica Gel H thin layers developed with n-butyl alcohol/acetic acid/water (5:2:3 v/v) and quantitated by zonal scraping of the plates followed by scintillation counting of the silica gel fractions. The identities of individual metabolites were confirmed by co-migration with standards in both the acidic chromatography system described above and a basic solvent system (ethanol/28% ammonium hydroxide, 4:1, v/v) as described previously (10).

Panothenate kinase activity was measured in COS-7 cells that had been incubated for 48 h at 37 °C following transfection with plasmid pmPanK1 or pcDNA3.1 vector. Transfected cell pellets were resuspended in 100 µl of hypotonic lysis buffer (10 mM Tris-Cl pH 7.5, 1 mM EDTA, 5 µg/ml leupeptin, 10 mM NaF) and left on ice for 30 min. Cells were then sonicated in a cup horn at maximum power three times for 30 s. Unbroken cells and cell debris were pelleted by centrifugation for 5 min at 5,000 × g. PanK activity was measured in aliquots of the cell extracts as described above.

Northern Blots-- A mouse multiple tissue Northern blot was purchased from CLONTECH and hybridized and washed according to the manufacturer's instructions. The blot was first hybridized with an 808-bp ³²P-labeled probe prepared from the digestion of cDNA clone AA030321 with AatII and NdeI which lies within the region common to both splice variants and which would signal the mRNAs encoding both mPanK1 isoforms. The mPanK1-specific 171-bp probe was prepared by PCR amplification using the mPanK1 cDNA clone W83049 as a template and the 5'-GAACGGGCTGCTGCACAAC-3' plus 5'-GTTCTTCCTCCCGGAGTCC-3' primer pair. The 187-bp mPanK1-specific probe was prepared by PCR amplification using the mPanK1 cDNA clone AI181055 as the template and the 5'-GGTAGACTGTAAAGGGTACC-3' plus 5'-GCTTTCTGCCATTTACAAGC-3' primer pair.

RT-PCR Detection of mPanK Isoforms-- RT-PCR was performed using mouse brain, heart, kidney, liver, or skeletal muscle poly(A)⁺ RNAs that were purchased from CLONTECH. The RNAs (200 ng) were used as template to synthesize the first strand cDNA using Superscript TMII reverse transcriptase (Life Technologies, Inc.) following the manufacturer's recommended procedure and using the gene-specific primer 5'-CGTAGGCAGTGTTAGAAGTTAAATAC-3' that was common to both mPanK1 and mPanK1 cDNAs. PCR amplification was carried out using 4 µl of the individual first-strand cDNAs as template together with a nested primer specific for the common region 5'-GCCGTGATATCCTTCGGTTC-3' (R289) plus either the mPanK1-specific primer 5'-GAACGGGCTGCTGCACAAC-3' (F35) or the mPanK1-specific primer 5'-GGTAGACTGTAAAGGGTACC-3' (A16). PCR reactions were performed in 20 µl with 35 thermocycles at 94 °C for 30 s for denaturation, 55 °C for 1 min for annealing, and 72 °C for 2 min extension. PCR products (8 µl) were separated by gel electrophoresis in 1.4% agarose.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation and Structure of mPanK1 cDNAs-- BLAST searches of the public expressed sequence tagged (EST) data base identified cDNA clones (GenBank^TM accession numbers AA030321 and AA014914 for mPanK1; AI181055 and AA105778 for mPanK1) with sequences homologous to the A. nidulans PanK (38) (Fig. 1). The clones were purchased, and the complete cDNA sequences were determined on both strands for each clone. Analysis of the sequence data revealed that the mRNAs were the products of the same gene, and the cDNAs were predicted to encode two protein isoforms, designated mPanK1 and mPanK1. The open reading frame encoding mPanK1 was preceded by an in-frame stop codon upstream from the first methionine. An in-frame stop codon in the 5'-sequence of the mPanK1 cDNA was not found, leaving open the possibility that the entire open reading frame had not been identified. Thus efforts were focused on characterization of the mPanK1 cDNA.

View larger version (71K): [in this window] [in a new window]   Fig. 1.   Alignment of the predicted amino acid sequences of mPanK1, mPanK1, aPanK, and yPanK. Amino acid residues identical to mPanK1 are highlighted. mPanK1 and mPanK1 are identical from residue 11 of mPanK1 to the end of the protein, designated as the common region. mPanK1, which has a different amino terminus from mPanK1, is predicted to be a splice variant of the mPanK1 mRNA. The mPanK1 predicted protein sequences are homologous to the predicted pantothenate kinase sequence from A. nidulans (aPanK) (38) and the predicted sequence of the YDR531W gene product (GenBankTM accession number 927798) identified in the S. cerevisiae genome (yPanK) (38).

The mPanK1 cDNA was predicted to encode a protein of 373 amino acids. The predicted molecular size of mPanK1 was 41,642 Da with a predicted isoelectric point of 6.03. The predicted amino acid sequence of mPanK1 included a methionine that aligned with the assigned translational start site of the mPanK1 cDNA (Fig. 1). The amino terminus of the  isoform differed from the  isoform from residues 2 to 10. Both isoforms were identical in amino acid sequence and in length from residues 11 to 373. The results of 3'-rapid amplification of cDNA ends on five different murine tissue poly(A)⁺ RNAs confirmed the presence of the polyadenylation signal (AATAAA) in the 3'-untranslated region followed by the poly(A)⁺ tail 22 bp downstream. These data confirmed the assignment of the stop codon after residue 373.

Tissue-specific Expression of mPanK1 and mPanK1 mRNA-- The relative abundance of mPanK1 mRNA expression in a variety of adult mouse tissues was addressed by Northern blot analysis (Fig. 2). Each lane contained 2 µg of poly(A)⁺ RNA isolated from the tissues indicated, and the blot was probed first with radiolabeled cDNA representing the coding sequence common to both mPanK1 and mPanK1. A 3.6-kb mRNA was detected at high levels in heart, liver, and kidney tissues, with lower levels in brain and skeletal muscle. A 3.1-kb mRNA was also detected at high levels in liver and kidney and to a lesser extent in testis. A larger RNA of approximately 5 kb was also signaled in liver. Radiolabeled probes specific for either the mPanK1 or the mPanK1 coding sequences were then hybridized sequentially to kidney poly(A)⁺ mRNA because this tissue expressed both mPanK1 mRNAs. These experiments revealed that the 3.6-kb mRNA corresponded to mPanK1 expression, whereas the smaller 3.1-kb mRNA corresponded to mPanK1 expression (Fig. 2). To verify the results obtained with the Northern blotting, RT-PCR was performed using poly(A)⁺ RNA isolated from various mouse tissues as template for the RT reaction. Isoform-specific primers corresponding to the 5'-ends of either the  or  cDNAs were paired with a primer corresponding to the mPanK1 common sequence in the PCR step. The mPanK1 message was confirmed to be in abundance in kidney and liver, whereas brain, heart, and skeletal muscle were found to express mPanK1 at a lower, but detectable, level (Fig. 3). The mPanK1 message was confirmed to be at high levels in heart, kidney, and liver, less in brain, and then at low but detectable levels in skeletal muscle (Fig. 3). These results were consistent with the conclusions derived from the Northern blot, where mPanK1 and mPanK1 mRNAs were abundant and present at about the same steady-state levels in kidney; mPanK1 expression was greater than mPanK1 in liver, and heart expressed primarily mPanK1. The enhanced sensitivity of RT-PCR enabled detection of relatively low expression of the two isoforms in skeletal muscle and in brain, where the expression of mPanK1 was slightly greater than that of mPanK1 (Figs. 2 and 3).

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Tissue-specific expression of mPanK1 isoforms. Left panel, multiple mouse tissue Northern blot signaled with a 32P-labeled probe generated from the common region of mPanK1 and mPanK1 cDNAs illustrates tissue-specific expression of two mRNAs that differ in size by approximately 600 bp. Right panel, identification of the mRNAs corresponding to mPanK1 and mPanK1 by hybridization of mouse kidney poly(A)+ mRNA with 32P-labeled probes specific for the mPanK1 or mPanK1 isoform. Kidney was selected because both mPanK1 mRNAs are abundantly expressed in this tissue. Molecular sizes were estimated from a calibration curve based on the standards indicated to the left of the figure, and the results from two independently prepared tissue blots were averaged. Details of the specific probes and blotting conditions are described under "Experimental Procedures."

View larger version (72K): [in this window] [in a new window]   Fig. 3.   RT-PCR analysis of mPanK1 and mPanK1 expression in mouse tissues. A, tissue distribution of mPanK1 evaluated using the F35 and R289 primer pair. B, tissue distribution of mPanK1 determined using the A16 and R289 primer pair. Primer R289 was a reverse primer from the common region of both mPanK1 cDNAs, whereas primer F35 was specific for the mPanK1 transcript, and A16 was specific for the mPanK1 transcript. C, control RT-PCR experiments: lane 1, primer F35 + template; lane 2, primer R289 + template; lane 3, primer A16 + template; lane 4, A16 + R289 without template. Details of the primers and PCR conditions are described under "Experimental Procedures."

Expression and Activity of mPanK1-- COS-7 cells were transfected with the expression plasmid pmPanK1 or pcDNA3.1 vector control, and 48 h later cell lysates were prepared. Pantothenate kinase enzymatic activity was significantly higher in the lysate from pmPanK1-transfected cells (Fig. 4). Endogenous pantothenate kinase was detected in the control lysates at protein concentrations of 50 µg and higher (data not shown) with an average specific activity of 3.24 ± 0.07 pmol/min/mg. In the experiment shown in Fig. 4, pantothenate kinase enzyme was overexpressed an estimated 250 times in the cells transfected with pmPanK1, indicating that a functional protein was encoded by the plasmid. Repeated transfections with the plasmid construct gave similar results.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   mPanK1 is a pantothenate kinase. COS-7 cells were transfected with either plasmid pcDNA3.1 (control) or the mPanK1 expression plasmid (pmPanK1). After 48 h, the cells were lysed, and the soluble extracts were evaluated for pantothenate kinase activity using the filter disc assay described under "Experimental Procedures." The data are representative of four independent experiments.

Effect of mPanK1 Expression on Pantothenate Metabolism-- Two sets of COS-7 cells were transfected with either the mPanK1 expression plasmid or the control vector. Cells within a set were pooled and re-plated into pantothenate-free Dulbecco's modified Eagle's medium plus dialyzed serum 24 h after transfection to avoid variation in transfection efficiency among replicate dishes. D-[1-¹⁴C]Pantothenate (2 µM) was added, and cells were radiolabeled for another 24 h at 37 °C and then harvested. Overexpression of mPanK1 was 919.9 ± 13.2 pmol/min/mg as determined by biochemical assay of an aliquot of the pooled cells from each set. The relative amounts of cellular pantothenate and its metabolites were compared in control cells and in cells overexpressing mPanK1 (Fig. 5). Pantothenate, which is the substrate for pantothenate kinase, constituted 43% of the total radiolabeled metabolite pool in control cells, whereas the product of the kinase, phosphopantothenate, was not detectable (Fig. 5A). These data indicated that the phosphorylation of pantothenate by the endogenous level of PanK was rate-limiting to the CoA biosynthetic pathway and was consistent with previous results obtained in bacteria (1) and mammals (18, 19). The data also demonstrated that the transport of pantothenate into the cell was not limiting, but rather cellular pantothenate accumulated prior to its phosphorylation by PanK. Neither 4'-phosphopantothenate nor 4'-phosphopantothenoylcysteine was detected, but phosphopantetheine was the second most abundant metabolite and constituted 35% of the total (Fig. 5A). These data suggested that the phosphopantetheine adenylyltransferase also catalyzed a slow step in the CoA biosynthetic pathway. The adenylyltransferase is three steps removed from PanK and converts 4'-phosphopantetheine into dephospho-CoA by addition of an adenine moiety. CoA was the third metabolite that was present in significant amounts, and it represented 22% of the total radiolabeled material (Fig. 5A), the smallest of the three major pools in the control cells.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of mPanK1 expression on the distribution of pantothenate-derived metabolites. Distribution of pantothenate-derived metabolites in cells transfected with the control vector, pcDNA3.1 (A), or the expression vector pmPanK1 (B). Cells were transfected, labeled with [14C]pantothenate for 24 h, and lysed, and the lysates were fractionated by thin layer chromatography on Silica Gel G layers developed in the acidic solvent system butanol/acetic acid/water (5:2:3, v/v). Radiolabeled metabolites were identified by co-migration with standards in both the acidic solvent (shown) and a basic solvent system (not shown) as described under "Experimental Procedures." The results are representative of two independent experiments.

In contrast, cells overexpressing mPanK1 did not accumulate pantothenate but readily metabolized it (Fig. 5B and Fig. 6). The total uptake of D-[1-¹⁴C]pantothenate also increased by about 4-fold in response to overexpression of mPanK1 (Fig. 6). The cellular radiolabeled pool was dominated by two metabolites, CoA (75%) and 4'-phosphopantetheine (25%). The CoA level was 13 times higher in cells with the PanK construct and, unlike in control cells, exceeded the level of 4'-phosphopantetheine. These data demonstrated that the expression level of PanK1 influenced both the uptake and metabolism of pantothenate and, most importantly, regulated the cellular CoA content. The data also indicated that whereas the phosphopantetheine adenylyltransferase may be a slow step in the pathway of CoA biosynthesis, possible regulation at this step was secondary to regulation by the PanK enzyme.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Quantitative changes in pantothenate metabolites in cells overexpressing mPanK1. COS-7 cells were transfected with either the control vector pcDNA3.1 or the expression plasmid pmPanK1 and labeled with [14C]pantothenate for 24 h. Intracellular 14C-labeled metabolites were extracted and quantitated by thin layer chromatography as described under "Experimental Procedures" (see also Fig. 5). The data shown were derived from a representative experiment that was replicated three times. In all experiments, mPanK1 overexpression resulted in at least a 10-fold increase of intracellular CoA levels. Data were obtained in the same experiment described in Fig. 5 and are representative of two independent experiments.

Biochemical Regulation of mPanK1 by CoA and Its Thioesters-- PanK activity from different sources is inhibited by CoA and/or CoA thioesters (15, 16, 18, 31-38). The possible role of biochemical feedback regulation of the mPanK1 activity was investigated using cell lysates prepared from COS-7 cells transfected with pmPanK1 48 h prior to harvest. We found that acetyl-CoA was the most potent regulator of the mPanK1 with an apparent IC₅₀ of about 20 µM (Fig. 7). Nearly complete inhibition of the overexpressed enzyme activity was achieved at 60 µM acetyl-CoA. Malonyl-CoA was also inhibitory but less effective, and the lowest activity that we observed in the biochemical assay was reduced to about 30% of control (Fig. 7). Surprisingly, unesterified CoA reproducibly stimulated enzyme activity and elevated the production of phosphopantothenate in the assay to about 140% of control values at concentrations 20 µM (Fig. 7). These data indicated that mPanK1 activity was regulated biochemically by the end products of the CoA biosynthetic pathway.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Influence of CoA, acetyl-CoA, and malonyl-CoA on the activity of mPanK1. Extracts from COS-7 cells transfected with the mPanK1 expression construct were prepared and assayed for pantothenate kinase activity in the presence of the indicated concentrations of CoA, acetyl-CoA, or malonyl-CoA as described under "Experimental Procedures." Endogenous PanK activity at this protein concentration (15 µg) from control cells was <0.2% of the rate in transfected cells. Values reported were averaged among quadruplicate samples. Results are representative of two experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The first identification of cDNAs encoding mammalian PanK has enabled us to test the hypothesis that PanK expression can govern the intracellular CoA level. The demonstration of a causal relationship between elevated mPanK1 protein and increased pantothenate uptake and metabolic incorporation into CoA (Figs. 5 and 6) validates the correlations previously observed between elevated tissue levels of PanK activity and increased CoA levels (29, 30, 34, 40). Enhanced expression of mPanK1 protein reduced the intracellular pantothenate level below detection limits, in contrast to control cells where pantothenate comprised a significant pool. Both observations are consistent with the idea that PanK activity governs the flux through the CoA biosynthetic pathway (17-19). Whereas the supply of extracellular pantothenate (2 µM) was sufficient to maintain a detectable pool of intracellular pantothenate in the control cultures, pantothenate may have been limiting to CoA production in the pmPanK1-transfected cultures (Figs. 5 and 6). These data suggest that tissues with higher PanK protein levels utilize pantothenate at a faster rate, in which case the exogenous pantothenate supply and transport would also play a role in determining the cellular CoA level. Heart is a tissue that expresses a high level of mPanK1 (Figs. 2 and 3), and earlier studies showed that increasing the exogenous pantothenate supply to heart tissue elevated cellular CoA content (41). Previous work also suggested that the supply of cysteine may be limiting to CoA biosynthesis in perfused heart tissue (19); however, our experiments did not find an accumulation of phosphopantothenate, an indicator of cysteine limitation (Fig. 8), either in control or transfected cultures.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Regulation of CoA biosynthesis. The vitamin pantothenate is transported into cells and phosphorylated by pantothenate kinase yielding 4'-phosphopantothenate. Condensation of 4'-phosphopantothenate with cysteine is followed by a decarboxylation reaction to yield 4'-phosphopantetheine. Next, the 5'-AMP moiety of ATP is added to form dephospho-CoA, which is subsequently phosphorylated on the 3'-hydroxyl to form CoA. CoA is then esterified to a variety of acyl moieties, the shortest being acetyl-CoA. The major metabolic intermediates in cells are indicated by the boxes. Pantothenate kinase is the major regulatory step controlling the pathway and is indicated by a large, bold triangle. Feedback regulation of pantothenate kinase activity is both positive by unesterified CoA and negative by acetyl-CoA. The phosphopantetheine adenylyltransferase is a secondary regulatory point as indicated by a smaller, bold triangle.

The metabolism of 4'-phosphopantetheine is a second control point in CoA homeostasis. CoA and 4'-phosphopantetheine are the two major metabolites of pantothenate (Figs. 5 and 8), and the increased level of PanK protein resulted in a 14-fold increase in the CoA level and a 3-fold increase in 4'-phosphopantetheine (Fig. 6). One possibility is that 4'-phosphopantetheine adenylyltransferase is a second regulatory enzyme in CoA biosynthesis (Fig. 8). This enzyme may also be limiting to CoA biosynthesis (Fig. 8), although regulation at the adenylyltransferase step is significantly less effective in controlling CoA levels than regulation at PanK. Alternatively, the elevated phosphopantetheine pool in mPanK1 overexpressing cells may arise from increased CoA degradation to phosphopantetheine. This mechanism is operative in E. coli when CoA levels are increased either by overexpression of bPanK (13) or in a coaA(Fr) mutant which expresses a bPanK resistant to CoA feedback inhibition (42). The excess phosphopantetheine exited the cell in E. coli, thus preventing re-synthesis of CoA (42). The compartmentation of the CoA biosynthetic pathway in mammals is an important, but poorly understood, aspect of metabolism. mPanK1 is clearly a soluble protein and is exclusively located in the high speed supernatant in the COS-7 cell extracts. However, mitochondria and peroxisomes are the two compartments with the highest concentrations of CoA and CoA thioesters (43, 44). One idea is that the last enzymes in the pathway are located inside the mitochondria (44, 45), and in this scenario, phosphopantetheine arising from CoA degradation may be expelled from these organelles into the cytosolic compartment where it cannot be further metabolized. Others report that mitochondria transport intact CoA into the matrix (46-48) implying that all of the pathway enzymes are cytosolic. Defining the cellular locations for synthesis and degradation will be important for understanding the mechanisms that regulation cellular CoA content.

Our results suggest that the levels of CoA and acetyl-CoA counteract each other in the regulation of PanK activity. Acetyl-CoA selectively inhibits the enzyme with an IC₅₀ value of about 20 µM, and these data are similar to those obtained for aPanK from A. nidulans (38). mPanK and aPanK have similarities in their protein sequences (Fig. 1), and there are several reports that acetyl-CoA is a potent inhibitor of mammalian PanK activity in cell extracts (17, 31-33, 35, 36, 40). Malonyl-CoA is less effective as an inhibitor of mPanK1, and surprisingly, unesterified CoA (16 µM) stimulates activity (Fig. 7). This finding is contrary to several reports of PanK inhibition by unesterified CoA in mammalian tissue extracts (17, 32, 33, 33-36, 40), although inhibition by CoA is always less potent than regulation by acetyl-CoA. CoA stimulation may be masked in cell extracts if the stimulation of mPanK1 by unesterified CoA is a unique property of this isoform. Compartmentation also plays a significant role in PanK regulation. Mitochondria and peroxisomes are the major reservoirs of CoA and acetyl-CoA, and thus the biochemical regulators are largely sequestered from the cytosolic PanK.

Cells modulate PanK expression to modify tissue CoA levels in response to diet and disease. PanK activity in heart decreased in response to glucose, pyruvate, fatty acids, or insulin (18), and hepatic concentrations of CoA increased in response to glucagon, starvation, high fat diet, hyperthyroidism, or diabetes (20, 22-25, 27, 28, 49). Treatment with clofibrate, glucocorticoids, or agents that increased cellular cyclic AMP resulted in elevated hepatic levels of both PanK activity and CoA (29, 30, 40), supporting a role for responsive PanK expression or modification in the determination of tissue CoA levels. However, it is not clear which PanK isoform is regulated by these hormones and drugs. In addition to mPanK1 and mPanK1, we have identified a third isozyme, mPanK2, that is encoded by a distinct gene.² Also, we have identified three distinct human isoforms.² Two molecular forms of PanK were previously identified in liver and were separated by isoelectric focusing with pI values of 5.1 or 5.7 (36). Acetyl-CoA inhibited both of the liver PanK isoforms; however, CoA only inhibited the activity with the pI of 5.7. Two molecular forms of PanK were previously characterized from spinach on the basis of their ion-exchange properties (37). It is not clear whether either of the two forms is the same as mPanK1 since the calculated isoelectric point of mPanK1 is 6.03 and it is inhibited by acetyl-CoA but stimulated by free CoA (Fig. 7). If the cDNA encoding mPanK1 corresponds to the complete protein, the calculated pI would be 5.79. Thus, the two isoforms noted in previous studies most likely arise from coexpression of mPanK1 and mPanK2 and/or post-translational modification of one or more PanKs. PanK phosphorylation may alter its biochemical properties, and the molecular reagents that evolve from this study will allow these and other questions to be addressed. PanK may be a feasible target for enforced regulation of cellular CoA levels by drugs.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants GM 45737 (to S. J.) and GM34496 (to C. O. R.), Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF200357.

^¶ To whom correspondence should be addressed: Biochemistry Dept., 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3494; Fax: 901-525-8025; E-mail: suzanne.jackowski@stjude.org.

² S. Jackowski, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: PanK, pantothenate kinase; CoA, coenzyme A, mPanK1, murine pantothenate kinase; aPanK, A. nidulans pantothenate kinase; yPanK, S. cerevisiae pantothenate kinase; bPanK, E. coli (bacterial) pantothenate kinase; PCR, polymerase chain reaction; EST, expressed sequence-tagged; bp, base pair; kb, kilobase pair.

	REFERENCES

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