Molecular Cloning of the DNA and Expression and Characterization of Rat Testes Fructose-6-phosphate,2-kinase:Fructose-2,6-bisphosphatase”

We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phos-phate,2-kinase:fructose-2,6-bisphosphatase. Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA frag- ment. A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a M, of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced M, of 54,023. Fru-6-P,Z-kinase:Fru-2,6-bis-phosphatase with the same M, 55,000 was also present in rat testis extract. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. Various kinetic constants of the expressed fructose 6-P,B-kinase were K P 6-p = 85 p~ and KkTP = 270 p ~ , and those of fructose 2,6- bisphosphatase were KF 2,6-p2 = 21 p~ and Kf’“ 6-p = 3.4 p ~ . The enzyme was phosphorylated by Fru-2,6[2- 32P]P2

Fru 6-P + ATP + Fru-2,6-P2 + ADP (1) Fru-2,6-P2 + H20 -Fru 6-P + Pi (2) Three major isozymic forms have been purified to apparent homogeneity from liver (9, lo), skeletal muscle (11,12), and heart (13), and their properties have been extensively char-* This work was supported by grants from the Department of Veterans Affairs Medical Center and Grant DK16194 from the National Institute of Diabetes, Digestive and Kidney Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" 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 GenBankTM/EMBL Data Bank with accession number(s) M64797.
acterized. In addition to these major isozymes, minor isozymic forms that are not well characterized occur in these tissues (14). Kinase activities of the major isozymes are similar (50-60 milliunits/mg) when assayed under identical conditions, but Fru-2,6-bisphosphatase activities are significantly different (33-154 milliunits/mg) (12,13). The cellular concentration of Fru-2,6-P2 is determined by the relative activities of the kinase and the phosphatase.The most important regulatory mechanism of these opposing activities is by phosphorylation and dephosphorylation of the enzymes. The liver enzyme is phosphorylated by CAMP-dependent protein kinase which results in inhibition of the kinase and activation of the phosphatase (15)(16)(17). Such a direct reciprocal relationship has been demonstrated in isolated hepatocytes treated with glucagon (18). This is consistent with the liver's role in inhibiting phosphofructokinase (and glycolysis) by decreasing the level of Fru-2,6-P2 in order to maintain glucose homeostasis under hormonal stimulation or starvation.
Rat skeletal muscle Fru-6-P, 2-kinase:Fru-2,6-bisphosphatase has been obtained in homogeneous form (12), and its tryptic peptide map is identical to that of rat liver isozyme except for two minor peptides (12). The muscle enzymes from pigeon breast (11) and rat (12) are not phosphorylated by CAMP protein kinase because the phosphorylation target Ser3' of the liver enzyme is replaced by Ala in the muscle enzyme (12). Consequently, the regulatory mechanism of the muscle enzyme is not understood.
In contrast to the liver enzyme, the heart isozyme is phosphorylated by both CAMP-dependent protein kinase and protein kinase C, both resulting in activation rather than inhibition of the kinase activity (19,ZO). Consistent with these in vitro results is the observation that epinephrine raises the Fru-2,6-Pz level in perfused heart (21), whereas the hormone decreases the Fru-2,6-P2 level in liver (23). The amino acid sequence of bovine heart enzyme has been determined (22), and the sequence shows that the phosphorylation sites for both protein kinases are located near each other in the Cterminal region (Ser466 and Thr47s). On the other hand, the phosphorylation site (by protein kinase A) of the rat liver enzyme is near the N terminus (24).
All the isozymes thus far studied extensively are from those tissues which metabolize a variety of substrates and therefore are not limited to glucose (or glycogen) for energy and other functions. Little is known about the nature and the types of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase in those glycolytic tissues such as brain or testis in which glucose is the primary source of energy. In this report we present the results of molecular cloning, DNA and amino acid sequence determinations, expression, and characterization of rat testis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase.

Materials
The X Zap I1 rat testis library was purchased from Stratagene (LaJolla, CA). The cDNA encoding human liver Fru-6-P,2-kinase:Fru-2,6-bisphosphatase was prepared as described (25). Restriction enzymes and bacteriophage T4 DNA ligase were from New England Biolabs. T 7 DNA polymerase and sequencing kit were purchased from Pharmacia LKB Biotechnology Inc. The pT7-7 RNA polymerase/promoter plasmid was a gift of Dr. Stan Tabor (Harvard Medical School). Rat liver Fru-6-P,2-kinase:Fru-2,6-bisphosphatase was purified according to the procedure of Sakakibara et al. (9). Fructose-2,6-[2-"P]P2 was prepared as described (6). Protein kinase C was a gift of Dr. Melanie H. Cobb (Department of Pharmacology, University of Texas Southwestern Medical Center a t Dallas). All other materials were reagent grade and obtained from commercial sources.

Methods
Isolation and DNA Sequence Determination of Rat Testis Fru-6-P,2-kinase:Fru-2,6-bisphosphutase-The h Zap I1 recombinant cDNA rat testis library plated on Escherichia coli XL1-Blue was screened initially with the cDNA probe encoding human liver Fru-6-P,2kinase:Fru-2,6-bisphosphatase (251, and later a DNA fragment of a rat testis clone RT6A (see Fig. 1) was used to rescreen the same library. The replicate nylon filters were prehybridized for 4-6 h a t 65 'C in 1% sodium dodecyl sulfate/l M NaCl containing fish DNA at 100 pg/ml and hybridized in the same solution containing a 32Plabeled probe at the same temperature overnight. The probes were radiolabeled with [w3*P]dCTP by random oligo-labeling (26). The filters were washed several times with 0.3 M NaCl, 30 mM sodium citrate, 0.2% sodium dodecyl sulfate a t 60 "C and subjected to autoradiography. For sequencing the pBluescript with the positive DNA inserts were recovered from the X Zap I1 phages by in vivo excision method using helper phage R408 (27), and the alkali-denatured double-stranded DNAs were sequenced by the dideoxy chain termination method (28) using universal, reverse, or custom primers.
RNA Blot Hybridization Analysis-Poly(A)+ RNA samples were prepared from fresh rat testis using an RNA isolation kit (Invitrogen, San Diego, CA). The RNAs were electrophoresed through formaldehyde/agarose gels (29), transferred to nitrocellulose filters (Schleicher and Schuell), and hybridized a t 65 "C in 0.5% sodium dodecyl sulfate, 1 M NaCl, and 0.1 M sodium citrate with the random-prime labeled RT6A cDNA, or RTOl4K DNA. RT014K DNA was prepared by polymerase chain reaction (30) using RT6S cDNA as a template and synthetic primer of 20 mer from nucleotides 1-140 in Fig. 2.
Assay Methods for Fru-6-P,S-kinase-The reaction mixture contained in a final volume of 0.1 ml, 100 mM Tris-HC1, pH 7.5, 2 mM dithiothreitol, 0.1 mM EDTA, 5 mM ATP, 1 mM Fru 6-P, 5 mM potassium phosphate, and 10 mM MgC12. The mixture was incubated at 30 "C and at timed intervals 10 pl-aliquots were transferred into 90 pl of 0.1 N NaOH, and the diluted solution was heated for 1 min at 90 "C to stop the reaction. Suitable aliquots of the heated reaction mixture were then assayed for Fru-2,6-P2 as described by Uyeda et al. (33). One unit of activity is defined as the amount of enzyme that catalyzes the formation of 1 pmol of Fru-2,6-P2 per min under these conditions.
Assay Methods for Fru-2,6-&isphosphatase-The reaction mixture contained in a final volume of 0.1 ml, 100 mM Tris-HCl, pH 7.5, 2 mM dithiothreitol, 5 mM MgCl,, 5 mM potassium phosphate, 50 p~ NADP, 0.4 unit of desalted glucose-6-P dehydrogenase, 1 unit of phosphoglucose isomerase, and 20 p~ Fru-2,6[2-"'P]P2 (8.2 X lo4 cpm/nmol) (34). The reaction was initiated with the addition of enzyme and incubated a t 30 "C. At timed intervals aliquots were transferred into 100 pl of 0.1 N NaOH, and the solution was heated at 80 "C for 2 min. H 2 0 (1 ml) was added to the heated reaction mixture, and the sample was adsorbed on a Dowex l-Cl-column (0.5 X 4 cm) equilibrated with 0.02 N NH4OH. The column was washed with 1 ml of 0.02 N NH,OH followed with 1 ml of 0.15 M NaCl in 0.02 N NH,OH. ["'PIPhosphate was eluted with 5 ml of the same solution; a 2-ml portion was diluted in 10 ml of Aquasol (Du Pont-New England Nuclear) and was counted in a scintillation counter. One unit of activity is defined as the amount of enzyme that catalyzes the formation of 1 pmol of phosphate per min under these conditions. Other Methods-Polyacrylamide slab gel electrophoresis was carried out in 10% acrylamide containing 0.1% sodium dodecyl sulfate according to the procedure of Laemmli (35). The gels were stained with Coomassie Blue and destained in methanol/acetic acid. Protein concentration was determined by the Bradford method (36) using bovine serum albumin as a standard.

RESULTS
Isolation and Characterization of cDNA Clones-Initial screening of the Stratagene rat testis library with the DNA encoding human liver Fru-6-P,2-kinase:Fru-2,6-bisphosphatase (25) yielded a positive clone containing a 1.5-kb insert designated RT6A. Rescreening the same library with RTGA DNA produced a clone containing a 0.65-kb insert, which was designated RT6S. The relationship between these two clones is shown in the restriction map in Fig. 1. The nucleotide sequence data ( Fig. 2) showed that the 3' end of RT6S (from nucleotide 1 to nucleotide 650) overlapped with the first 402 nucleotides of the 5' end of RTGA. RT6S contained 34 nucleotides of 5'-untranslated sequence followed by an initiation codon. This initiation site and the surrounding nucleotide sequence had the general consensus structure of a eukaryotic initiation site. The composite nucleotide sequences of these clones was 1773 nucleotides in length and, in addition to 5'and 3"noncoding sequences, contains an open reading frame of 1404 nucleotides. The deduced amino acid sequence of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase of rat testis is shown in Fig. 2. The specified protein contained 468 amino acid residues and the calculated molecular weight was 54,023. mRNA in Rat Testis-To analyze Fru-G-P,2-kinase:Fru-2,6-bisphosphatase isozyme distribution in rat testis, Northern blot analyses of mRNAs isolated from testis were performed using two DNA probes. A DNA RT014K (nucleotides 1-140; Fig. 2), specific for mRNA of the testis enzyme, and a RTGA DNA (Fig. l), having sequence homology with both liver and heart type enzyme DNAs (22,25), were labeled with random priming and used as probes to detect complementary mRNA sequences under high stringency conditions. The Northern blot analysis detected two species of mRNA in rat testis (Fig. 3). A 3.3-kb mRNA hybridized with the RT6A probe (lane I ) but not with RT014 (lune 2 ) . However, a 2-kb mRNA hybridized with both probes indicating that the 2-kb mRNA was specific for rat testis enzyme. Furthermore, judging from the intensity of the bands, the 2-kb mRNA was considerably more abundant than the 3.3-kb mRNA.

Q N V D I S R P S E E A L V T V P
The full length cDNA of the Fru-6-P,2-kinase:Fru-2,6-bisphosphatase was amplified and digested with BglII and PstI and then ligated with synthetic oligoDNA having NdeI-BgZII cohesive ends, which was designed to match the reading frame of the bacteriophage T7 direct-expression system (31). Insertion of this fragment into the NdeI-PstI-digested pT7-7 yielded a construct, pT7-7/RT2K, that contained the full length Fru amide gel electrophoresis under denaturing conditions. As shown in Fig. 5 the time course for the expression of the testis enzyme indicated that the enzyme (arrow) was expressed rapidly and appeared to reach the maximum value in 2 h. The corresponding protein band was absent in the zero time sample. On the basis of the specific catalytic activity of the purified testis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase, the transformant of E. coli carrying the DNA produced the enzyme as approximately 3% of the soluble protein in cells harvested at 2.5 h after induction.  Fig. 5; lane 2, the expressed rat test,is enzyme; lane 3, rat liver enzyme.

Comparison of the kinetic constants of rat testis (expressed), liver, muscle, and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase
The values for testis enzymes were the average f S.D. of three to six determinations. The values for liver, muscle, and heart enzymes were from Refs. 12 and 13, respectively.

Purification of the Expressed Fru-6-P,2-kinase:Fru-2,6-bis-
phosphatase-A 2-liter culture of BL21(DE3) containingpT7-7/RT2K was harvested by centrifugation after 2.5 h of induction. The pellet was suspended in 60 ml of 50 mM Tris/ phosphate buffer, pH 8.0,2 mM dithiothreitol, 0.5 mM EDTA, 0.5 mM EGTA, 1% polyethylene glycol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 1 pg/ml leupeptin, and 1 mg/ml of lysozyme, and the suspension was kept on ice for 30 min. The cell lysate was centrifuged for 30 min, and the supernatant solution was applied to a DEAE-cellulose (1 X 7.5 cm) column (DE52, Whatman, Hillsboro, OR) which had been equilibrated with the same buffer mixture without lysozyme. After washing the column with 0.12 M KC1 in the buffer mixture, the enzyme was eluted with 0.3 M KCl. The eluted enzyme was diluted 2-fold with 50 mM HEPES, pH 7.5,5 mM potassium phosphate, 2 mM dithiothreitol, 0.5 mM EDTA, 0.5 mM EGTA, 1% polyethylene glycol, 5% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, and 1 pg/ ml leupeptin (termed "solution A ) and the enzyme solution was adsorbed on a Blue-Sepharose (1 X 5 cm) column (Pharmacia LKB Biotechnology Inc.). The column was washed with 20 ml of 0.4 M KC1 in solution A and eluted with 1.5 M KC1 in solution A. The fraction containing the activity was concentrated with Centricon-30 (Amicon) and subjected to molecular seive filtration on a Superose 12 column (Pharmacia) which had been equilibrated with solution A. The purified enzyme was concentrated with Centricon-30 again and stored frozen a t -80 "C. A typical example of this purification procedure is summarized in Table I.
The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis under denaturing conditions (Fig. 6).
Physical of the active enzyme was estimated as 11 X 10" indicating that the enzyme was a dimer.
Kinetic Properties of the Expressed Enzyme- Table I1 compares kinetic constants of the expressed rat testis enzyme with those of rat liver (9), rat muscle ( E ) , and bovine heart (13) enzymes. The expressed rat testis Fru-B-P,2-kinase had approximately 1.5 x higher activity than other kinases, but Fru-2,6-bisphosphatase activity of the testis enzyme was the lowest among those isozymes. In general, the kinetic constants of the testis enzymes were similar to those of bovine heart isozyme. Phosphorylation of Fru-2,6-bisphosphatase with Fru-2,6-P2-The rate of phosphorylation of Fru-2,6-bisphosphatase by its substrate [2-:'2P]Fru-2,6-P2 and the rate of dephosphorylation of ""P-phospho enzyme were similar to the rates of rat liver enzyme (Fig. 7). The extent of phosphate incorporation, however, was lower (0.42 mol of P incorporated per mol subunit) than that (0.55 mol of P per mol) of the liver Fru-2,6-bisphosphatase.
T o confirm the existence of the same enzyme in rat testis in uiuo, Fru-6-P,2-kinase:Fru-2,6-bisphosphatase in tissue extracts was phosphorylated with [2-'"P]Fru-2,6-P2 and subjected to polyacrylamide gel electrophoresis along with expressed phosphorylated enzyme. The results shown in Fig. 8 demonstrate that a '"P-labeled enzyme with identical M, was  detectable in testis extract, suggesting that the enzyme is present in uiuo.
Phosphorylation by Protein Kinase C-The rat testis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase could not be phosphorylated by CAMP-dependent protein kinase, unlike the rat liver or bovine heart isozymes. However, the testis enzyme was phosphorylated by protein kinase C and the phosphorylation required the presence of phosphatidyl serine, Ca2+, and diolein (data not shown). The phosphorylation site was not determined, but based on a computer search for a potential site for protein kinase C, the likely target serine was localized in serine 28 or threonine 443.

DISCUSSION
The cDNA clone for rat testis Fru-6-P,2-kinase:Fru-2,6bisphosphatase reported herein encoded an enzymatically active form of the enzyme. Our previous results, based on ionexchange chromatography and immunoreaction, showed the existence of a heart-type isozyme and an unidentified form of the isozyme which is more abundant in rat testis. The present results indicate that the cloned enzyme is the testis-specific isozyme. This conclusion was reached based on the following observations: (a) The Northern blot analysis (Fig. 3) demonstrated that the 2.0-kb mRNA specified the testis enzyme as determined by the testis enzyme DNA probe. ( b ) The expressed enzyme showed the same characteristics as the major enzyme present in rat testis extract. Earlier we detected a second isozyme in testis which appears to be a heart-type isozyme, since the chromatographic behavior and immunoreaction are similar to those of a heart-type isozyme. ' Various kinetic constants of the expressed testis enzyme are different from other major isozymes of liver, skeletal muscle, and heart isozymes. However, some of the isozymes have been reported to have comparable or higher kinase activity than the testis enzyme. The specific activities of those preparations range from 100 to 180 milliunits/mg for beef heart (13,39,40) and pigeon muscle (41). However, some of these differences may be due to different assay conditions and to different isozymic forms in the tissue. For example, we have shown that the major isozyme (Mr 58,000) in bovine heart has the specific kinase activity of 61 milliunits/mg, but the minor isozyme (M, 54,000) shows 175 milliunits/mg. It is interesting that the testis enzyme has the highest Fru-6-P,2kinase activity and the lowest Fru-2,6-bisphosphatase activity. Since the Fru-2,6-P2 level in cells is controlled by the relative activities of kinase/phosphatase, the high ratio of these two activities in testis may reflect the importance of Fru-2,6-P2 in a glycolytic tissue such as testis.
One of the important regulatory mechanisms of liver and heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase is phosphorylation and dephosphorylation as discussed in the introduction. I t is possible that the testis enzyme also is regulated by the same mechanism. The testis enzyme was not phosphorylated by CAMP-dependent protein kinase, unlike the liver and heart isozymes, but similar to rat skeletal muscle enzyme. However, it was phosphorylated by protein kinase C, and in this respect the enzyme was similar to the bovine heart isozyme. The significance of the phosphorylation of the testis enzyme is not certain. The testis enzyme contained three potential phosphorylation sites for protein kinase C, namely Ser', Ser28, and Thr4". Because of the opposite effects on the enzyme activities depending upon the location of the phosphorylation site(s), i.e. N or C terminus of the enzyme (19), it is of great interest to distinguish these sites in the testis enzyme. Currently we are ascertaining the location of the phosphorylation site and determining its effect on the activities of the bifunctional enzyme. These studies may elucidate the regulatory mechanism of phosphofructokinase and glycolysis in testis tissue.
A comparison of the amino acid sequence of rat testis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with those of rat liver (42,43), rat heart (this paper), and bovine heart (22) enzymes is presented in Fig. 9. T o maximize the alignment of these sequences one amino acid gap had to be added to the testis and two gaps to the liver enzyme sequences. During the course of this work, we have also isolated from the same library a cDNA clone which encoded for rat heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase. The deduced amino acid sequence of this clone corresponded to Pro'* to the C terminus of the sequence of the bovine heart enzyme, and those sequences of the heart enzymes were 94% identical. The rat testis enzyme showed 74,67, and 64% similarity with rat liver, rat heart, and bovine heart enzymes, respectively. The major differences among these sequences were in the N and C termini where the regulatory phosphorylation sites are 10cated. Although the C termini of the testis and the liver ' R. Sakakibara and K. Uyeda, unpublished results.

Testis
-ASPRELTQNPLKKIWHPYSNGRPAUL&SQR enzymes are similar (13 out of 18 amino acids from Val4" to the C terminus of the testis enzyme), their N-terminal sequences are different. On the other hand, both termini of the heart isozymes are completely different from those of the testis and the liver isozymes. It is noteworthy that the Cterminal residues of bovine and rat heart enzymes containing the phosphorylation sites for CAMP-dependent protein kinase and protein kinase C (22) are nearly identical. Beyond these terminal regions there are considerable numbers of amino acid sequences that are highly conserved (indicated in blocks). It is interesting that the C-terminal halves of these enzyme molecules (Fru-2,6-bisphosphatase domain) beginning from Leu'" of the testis enzyme are strikingly similar. Moreover, most of the differing amino acids are conservative substitutions. Among the highly conserved sequences, it is not surprising to find various substrate and product binding sites. The characteristic nucleotide binding site (44) is Gly-Leu-Pro-Ala-Arg-Gly-Lys-Thr (residues 45-52 of the testis enzyme sequence) in these enzymes. The Fru-6-P binding sites, as determined by affinity labeling experiments using N-bromoacetylethanolamine-P, are Cys'07 of the liver enzyme and the corresponding residues, Cys'05 and Cyslg6, of the heart enzymes (45). The amino acid sequences surrounding these Cys residues are well conserved. We also described earlier (37) conditions for the modification of the liver enzyme with pyridoxal-P which results in selective modification of two Lys residues involved in Fru-2,6-P2 and ATP binding. These Lys residues, Lys117 and Lys3"j, of the liver enzyme are also conserved in both the testis and heart enzymes. It is noteworthy that all of these substrate binding sites are for Fru-6-P,2kinase and localized in the N-terminal half of the molecule. On the other hand, the binding sites of Fru-2,6-Pz, the substrate, and for Fru 6-P, the inhibitor of Fru-2,6-bisphosphatase, have not been identified. Apparently these reagents that react with the same sugar phosphate binding sites of Fru-6-P,2-kinase do not react with Fru-2,6-bisphosphatase sites, which may suggest that the active site is buried. The only site identified thus far is the active site, His'". The phospho-His258 as the reaction intermediate of Fru-2,6-bisphosphatase (38) and its neighboring amino acids (Ile253, G1uZfi2 of the liver enzyme) are also identical in all these enzymes. Thus, the additional ligand binding sites of Fru-2,6-bisphosphatase as well as other sites of Fru-B-P,2-kinase need to be elucidated.