Molecular Cloning of the DNA and Expression and Characterization of. Rat Testes Fructose-6-phosphate,2-kinase:Fructose-2,6- bisphosphataseâ. (Received for ...
THEJOURNALOF BIOLOGICAL CHEMISTRY
Vol. 266, No. 24, Issue of August 25, pp. 15764-15770,1991 Printed in U.S.A.
Molecular Cloningof the DNA and Expression and Characterizationof Rat Testes Fructose-6-phosphate,2-kinase:Fructose-2,6bisphosphatase” (Received for publication, March 25, 1991)
Junichiro Sakata, Yumiko Abe,and Kosaku Uyeda From the Preclinical Science Unit, Department of Veterans Affairs Medical Center, and the Department of Biochemistry, University of Texas Southwestern Medical Center,Dallas, Texas 75216
We have isolated and sequenced two overlapping acterized. In addition to these major isozymes, minor isozymic cDNA fragments which could encode the complete forms that are not well characterized occur in these tissues amino acid sequence of rat testis fructose-6-phos- (14). Kinase activities of the major isozymes are similar (50phate,2-kinase:fructose-2,6-bisphosphatase. Northern 60 milliunits/mg) when assayed under identical conditions, blot analysis revealed that the major 2-kilobase mRNA but Fru-2,6-bisphosphatase activities aresignificantly differisolated from rat testis hybridized with a cDNA frag- ent (33-154 milliunits/mg) (12, 13). The cellular concentrament. A full length cDNA, which encoded a protein of tion of Fru-2,6-P2 is determined by the relative activities of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to the kinase and the phosphatase.Themost important regulahomogeneity, showed a M, of 55,000 by gel electro- tory mechanismof these opposing activities isby phosphorylphoresis under denaturing conditions, compared to the ation and dephosphorylation of the enzymes. The liver endeduced M, of 54,023. Fru-6-P,Z-kinase:Fru-2,6-bis-zyme is phosphorylated by CAMP-dependent protein kinase of the phosphatase with the same M, 55,000 was alsopresent which results in inhibitionof the kinase and activation in rat testis extract.The active enzyme was a dimer as phosphatase (15-17). Such a direct reciprocal relationship has judgedbymolecular sieve filtration. The expressed been demonstrated in isolated hepatocytes treated with gluliver’s role in inhibiting enzyme was bifunctional with specific activities of 90 cagon (18).This is consistent with the and 22 milliunits/mg of the kinase and the phosphatase phosphofructokinase (and glycolysis) by decreasing the level activities, respectively. Various kinetic constants of of Fru-2,6-P2 in order to maintain glucose homeostasis under the expressed fructose 6-P,B-kinase wereK P 6-p = 8 5 hormonal stimulation or starvation. p~ and KkTP= 270 p ~ and , those of fructose 2,6Rat skeletalmuscle Fru-6-P, 2-kinase:Fru-2,6-bisphosphabisphosphatase were KF 2,6-p2 = 21 p~ and Kf’“6-p = tase has been obtained in homogeneous form (12), and its 3.4 p ~ The . enzyme was phosphorylatedby Fru-2,6[2- tryptic peptide map is identical to that of rat liver isozyme 32P]P2and also by protein kinase C, but not by CAMP- except for two minor peptides (12). The muscle enzymes from dependent protein kinase, which is in contrast to the pigeon breast (11) and rat (12) are not phosphorylated by liver and heart isozymes. CAMPproteinkinase because thephosphorylationtarget Ser3’of the liver enzyme is replaced by Ala in the muscle enzyme (12). Consequently, theregulatory mechanism of the Fructose-2,6-P2 is the most potent activator of phospho- muscle enzyme is not understood. In contrast to theliver enzyme, the heart isozyme is phosfructokinase, a key regulatory enzyme of glycolysis (see Ref. 1 for review). The synthesis and degradation of Fru-2,6-P21 phorylated by both CAMP-dependent protein kinase and proare catalyzed by a bifunctional enzyme Fru-6-P,P-kinase and tein kinase C, both resulting in activation rather than inhiFru-2,6-bisphosphatase (Equations 1 and 2) (2-8) as follows. bition of the kinase activity(19,ZO). Consistent with these in raises the Fru 6-P + ATP + Fru-2,6-P2 + ADP (1) vitro results is the observation that epinephrine Fru-2,6-Pz level in perfused heart (21), whereas the hormone Fru-2,6-P2 + H 2 0 Fru 6-P + Pi (2) decreases the Fru-2,6-P2 level in liver (23). The amino acid Three major isozymic forms have been purified to apparent sequence of bovine heart enzyme has been determined (22), homogeneity from liver (9, lo), skeletal muscle (11, 12), and and the sequence shows that the phosphorylation sites for heart (13), and their properties have been extensively char- both protein kinases are located near each other in the Cterminal region (Ser466and Thr47s). On the other hand, the A) of the rat liver * This work was supported by grants from the Department of phosphorylation site (by protein kinase Veterans Affairs Medical Center and Grant DK16194 from the Na- enzyme is near theN terminus (24). tional Instituteof Diabetes, Digestive and KidneyDiseases. The costs All the isozymes thus far studied extensively are from those of publication of this article were defrayed in part by the paymentof tissues which metabolize avariety of substrates and therefore page charges. This article must therefore be hereby marked “aduertisement” in accordance with18U.S.C. Section 1734 solely to indicate are not limited toglucose (or glycogen) for energy and other functions. Little is known about the nature and the typesof this fact. The nucleotide sequence(s)reported in this paper has been submittedFru-6-P,2-kinase:Fru-2,6-bisphosphatase in those glycolytic totheGenBankTM/EMBLDataBankwith accession number(s) tissues such as brain or testis which in glucose is the primary M64797. source of energy. In this report we present the results of ’ The abbreviations used are:Fru-2,6-P2,fructose-2,6-bisphosphatase;EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; molecular cloning, DNA and amino acid sequence determinations, expression, and characterizationof rat testis Fru-6HEPES,4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid kb, kilobase(s). P,2-kinase:Fru-2,6-bisphosphatase.
-
15764
Testis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase
15765
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 Materials mixture, and the samplewas adsorbed on a Dowex l-Cl- column (0.5 The X Zap I1 rat testis library was purchased from Stratagene X 4 cm) equilibrated with 0.02 N NH4OH. The column was washed (LaJolla, CA). The cDNAencodinghuman liver Fru-6-P,2-ki- with 1 ml of 0.02 N NH,OH followed with 1 ml of 0.15 M NaCl in nase:Fru-2,6-bisphosphatasewas prepared as described (25). Restric- 0.02 N NH,OH. ["'PIPhosphate was eluted with 5 ml of the same tion enzymes andbacteriophageT4 DNAligasewere from New solution; a 2-ml portion was diluted in 10 ml of Aquasol (Du PontEngland Biolabs. T 7 DNA polymerase and sequencing kit were pur- New England Nuclear) and was counted in a scintillation counter. chased from Pharmacia LKB Biotechnology Inc. The pT7-7 RNA One unitof activity is defined as the amount of enzyme that catalyzes polymerase/promoter plasmid was a gift of Dr. Stan Tabor (Harvard the formationof 1 pmol of phosphate per min under these conditions. Medical School). Rat liver Fru-6-P,2-kinase:Fru-2,6-bisphosphatase Other Methods-Polyacrylamide slab gel electrophoresis was carwas purifiedaccording to the procedure of Sakakibara et al. (9). ried out in 10% acrylamide containing 0.1% sodium dodecyl sulfate Fructose-2,6-[2-"P]P2 was prepared as described (6). Protein kinase according to the procedure of Laemmli (35). The gels were stained C was a gift of Dr. Melanie H. Cobb (Department of Pharmacology, with Coomassie Blue and destained in methanol/aceticacid. Protein University of Texas Southwestern Medical Center a t Dallas). All concentration was determined by the Bradford method (36) using other materials were reagent grade and obtained from commercial bovine serum albumin asa standard. sources. EXPERIMENTALPROCEDURES
RESULTS
Methods Isolation and DNA Sequence Determination of Rat Testis Fru-6P,2-kinase:Fru-2,6-bisphosphutase-The h Zap I1 recombinant cDNA rat testis library plated on Escherichia coli XL1-Blue was screened initiallywiththecDNAprobeencodinghuman liver Fru-6-P,2kinase:Fru-2,6-bisphosphatase(251, and later a DNA fragment of a rat testis clone RT6A (see Fig. 1) wasused 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 thepositive DNA inserts were recovered from the X Zap I1 phages by in vivo excision method usinghelperphageR408 (27),andthealkali-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 testisusing an RNA isolation kit (Invitrogen, San Diego, CA). The RNAs were electrophoresed through formaldehyde/agarose gels (29),transferred tonitrocellulose filters (Schleicher and Schuell), andhybridized 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 DNAwas prepared by polymerase chain reaction (30) using RT6S cDNAas a template and synthetic primer of 20 mer from nucleotides 1-140 in Fig. 2. Expression of theRat TestisBifunctional EnzymeinE. coligene was Expression of the Fru-6-P,2-kinase:Fru-2,6-bisphosphatase directed by the bacteriophageT 7 expression system as described (31, 32). The expression plasmid pT7-7/RT2K was transformed into E. coli BL21(DE3), where the gene for T 7 RNA polymerase is located in thechromosome under control of the inducible LacUV5 promoter. The culture was grown at 37 "C in 2% tryptone, 1% yeast extract, 0.5% NaC1, 0.2% glycerol, 50 mM potassium phosphate, p H 7.5, and 50 pg/ml ampicillin. When the culture ODs9,, reached 0.5, T 7 polymerase was induced by adding 0.5 mM isopropyl-P-D-thiogalactoside. Preparation of Rat Testis Tissue Extract-Rat testis extract conactivity was pretaining theFru-6-P,2-kinase:Fru-2,6-bisphosphatase pared and further purified with a Blue-Sepharose columnas described (14). 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 10mM 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 amountof 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-Pdehydrogenase, 1 unit of phosphoglucoseisomerase, and 20 p~ Fru-2,6[2-"'P]P2 (8.2 X lo4 cpm/nmol) (34). The reaction was initiatedwiththeaddition of
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 produceda clone containinga 0.65-kb insert, which was designated RT6S. The relationshipbetween these two clones is shown in the restriction map in Fig. 1. The nucleotide sequence data (Fig. 2) showed that the3' end of RT6S (from nucleotide 1 to nucleotide 650) overlapped with the first 402 nucleotides of the 5' end of RTGA. RT6S contained34 nucleotides of 5'-untranslated sequence followed by an initiation codon. This initiation site and the surrounding nucleotide sequence had the general consensus structureof 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 Fru6-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 analyzeFru-G-P,2-kinase:Fru2,6-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 typeenzyme 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 detectedtwo 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 the3.3-kb mRNA. Subcloning and Expression-The overall procedure of constructing the plasmid for directexpression of rat testis Fru6-P,2-kinase:Fru-2,6-bisphosphatase is summarized in Fig. 4. Bgl II EcoRI
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5'
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ACCl A
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RTGS RTGA
05Kb
FIG.1. Schematic representation of a Fru-6-P,B-kinase: Fru-2,6-bisphosphatase cDNA from rat testis. The bar indicates the coding region; lines under bar show the DNA contained in the fragments RT6S and RT6A. Restriction sites are indicated on the bar.
TestisFru-6-P,2-kinase:Fru-2,6-bisphosphatase
15766
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661
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FIG. 3. Northern blot analysis of poly(A)+ RNA isolated from rat testis. Poly(A)+ RNAs (10 pg) from rat testis were separated by electrophoresis and hybridized with'"P-labeled RT6A cDNA having homology with mRNAs of liver and heart type enzymes (22, 25) (lane I ) and RTOl4k DNA (nucleotides 1-140,Fig. 2)specific for mRNA of testis enzyme (lane 2). The migration of RNA markers (7.46-1.35 kb) is indicated.
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FIG. 2. Nucleotide and deduced amino acid sequences. The nucleotidesequencebeginswith the 5' end of the DNA insert; nucleotides representing the proposed coding region are capitalized. pT7.7tUTZK Number.? on theleft refer to thenucleotide sequence. Fragment RT6S contained bases 1 to 651, and fragment RT6A containedbases 248 to FIG. 4. Schematic description of the construction of an 1739. Numbers on the right refer to thededuced amino acid sequence expression vector, pT7-7/RT2K, containing the full length given in one-letter code below the nucleotide sequence. testis Fru-6-P 2-kinase:Fru-2,6-bisphosphatasecDNA.pBluescript/RTZK contains the full coding region of the new isozyme. The T o synthesize the full length cDNA encoding the testis Fru- syntheticfragment coding amino acids 1-15 was formed by the 6-P,2-kinase:Fru-2,6-bisphosphatase, pBluescript-containing annealing of two complementary oligonucleotides: 5"TATGGCGTCfragment RT6S was digested with S a d and PstI. ThecDNA CCCACGGGAACTGACCCAGAATCCCCTGAAGAA-3' and 5'-
fragment RT6A was digested with the same restriction enzymes, isolated, and introduced into the Sad-PstI-digested pBluescript/RTGS("pBluescript/RT2K"). The full length was cDNA of the Fru-6-P,2-kinase:Fru-2,6-bisphosphatase 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 T 7 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-6P,2-kinase:Fru-2,6-bisphosphatase cDNA attached directly to the translation initiation codon of pT7-7. The constructed cDNA was validated by dideoxynucleotidesequencing and restriction analysis. This construct was stable in E. coli. The proteins expressed in these cells were analyzed by polyacryl-
GATCTTCTTCAGGGGATTCTGGGTCAGTTCCCGTGGGGACG C C A - 3 '. The bars show rat testis Fru-6-P,2-kinase:Fru-2,6-
bisphosphatase cDNA. ATG and TGA indicatetheinitiatorand terminator codons, respectively, of the enzyme. 610,T7 RNA polymerase promoter.
amide gel electrophoresis under denaturing conditions. As shown in Fig. 5 the timecourse forthe expression of the testis enzyme indicatedthatthe enzyme (arrow) was expressed rapidly and appeared to reach the maximum value in 2 h.The corresponding protein bandwas absent in thezero time sample. On the basis of the specific catalyticactivity of the the purified testis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase, 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.
Testis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase 1
2
3
4
15767 1
5
2
3
FIG. 6. Polyacrylamide gel electrophoresis in sodium dodeFIG. 5. Induction of the Fru-6-P,2-kinase:Fru-2,6-bisphos- cy1 sulfate of the purified expressed rat testis and rat liver phatase. The lysogen BL21(DE3)carrying pT7-7/RT2K was grown Fru-6-P,2-kinase:Fru-2,6-bisphosphatase.Lone I , standard and protein synthesis was induced with 0.5 mM isopropyl @+-thio- protein markers as in Fig. 5; lane 2, the expressed rat test,isenzyme; galactoside. At 0 (lane 2),0.5 (lane 3), l (lane 4 ) and 2 h (lane 5) lane 3,rat liver enzyme. after induction, 1 ml of the culture was centrifuged and processed for polyacrylamide gel electrophoresis as described under "Methods." TABLEI1 Ixne I contains standard marker proteins (fromtop): phosphorylase Comparison of the kinetic constants of rat testis (expressed), liver, b (MW 92500), bovine serum albumin (MW 68000), ovalbumin(MW muscle, and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase 45000), carbonic anhydrase(MW 31000) and soy bean trypsin inhibThe values for testis enzymes were the average f S.D. of three to itor (MW 21500). The arrow indicates the position of the expressed six determinations. The values for liver, muscle, and heart enzymes protein (MW 55000). were from Refs. 12 and 13, respectively. TABLEI Purification of expressed Fru-6-P,2-Kinase:Fru-2,6-bisphosphatase
Rat
Fru-B-P.2-kinsse
rnl
Lysate
DEAE-cellulose Blue-Sepharose SuDerose 12 " mu, milliunits.
rng
60
66
40
17
20 1.6
3.2 0.5
Testis
Fru-g-P,P-kinase K;""." (pM) KkTP( P M )
mLI"
rnlJ/rng
%
200 156 107
3.0 9.2 33.4
100
V,,, (milliunits/mg)
78
Fru-2,6-bisphosphatase
46
92
54
23
Kp2.6.1'2
KrnL6.P
(PM)
(*M)
Vmnx(milliunits/mg)
85 2 5 2 7 0 2 43 90 f 5 21 f 4.7 3.4 -c 0.2 22 f 3.3
Bovine
Liver
Muscle
heart
16
310
56 48
74 260
57
66
61
>I
3.5 46
0.4
12.5 154
40 11
33 "
Purification of the Expressed Fru-6-P,2-kinase:Fru-2,6-bispressed rattestis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase phosphatase-A 2-liter cultureof BL21(DE3) containingpT7- should contain 468 amino acids, and the calculated M,of the 7/RT2K was harvested by centrifugation after2.5 h of induc- subunit of rat testis Fru-6-P, 2-kinase:Fru-2, 6-bisphosphation. The pellet was suspended in 60ml of 50 mM Tris/ tase was 54,023. The polyacrylamide gel electrophoresis under phosphate buffer, pH 8.0,2mM dithiothreitol, 0.5 mM EDTA, denaturing conditions showed that the expressed testis en0.5 mM EGTA, 1%polyethylene glycol, 0.5 mM phenylmeth- zyme had the subunit M, as 55,000 (Fig. 6). Based on the ylsulfonyl fluoride, 2mM benzamidine, 1pg/ml leupeptin, and elution position of the enzyme from a Superose column, M, 1 mg/ml of lysozyme, and the suspensionwas kept on ice for of the active enzyme was estimated as 11 X 10" indicating 30 min. The cell lysate was centrifuged for 30 min, and the that theenzyme was a dimer. supernatant solution was applied to a DEAE-cellulose (1 X Kinetic Properties of the Expressed Enzyme-Table I1 com7.5 cm) column (DE52, Whatman, Hillsboro, OR) which had pares kinetic constants of the expressed rat testis enzyme been equilibrated with the samebuffer mixture without lyso- with those of rat liver (9), rat muscle ( E ) , and bovine heart zyme. After washingthe column with 0.12 M KC1 in thebuffer (13) enzymes. The expressed rat testis Fru-B-P,2-kinase had mixture, the enzyme was eluted with 0.3 M KCl. The eluted approximately 1.5 x higher activity than other kinases, but enzyme was diluted 2-fold with 50 mM HEPES, pH 7.5,5mM Fru-2,6-bisphosphatase activityof the testis enzyme was the potassium phosphate,2 mM dithiothreitol, 0.5 mM EDTA, 0.5 lowest among thoseisozymes. In general, the kinetic constants mM EGTA, 1% polyethylene glycol, 5% glycerol, 0.5 mM of the testis enzymes were similar to those of bovine heart phenylmethylsulfonyl fluoride, 2 mM benzamidine, and 1pg/ isozyme. ml leupeptin (termed "solution A ) and the enzyme solution Phosphorylation of Fru-2,6-bisphosphatase with Fru-2,6was adsorbed on a Blue-Sepharose (1 X 5 cm) column (PharP2-The rate of phosphorylation of Fru-2,6-bisphosphatase macia LKB Biotechnology Inc.). The columnwaswashed with 20 ml of 0.4 M KC1 in solution A and eluted with 1.5 M by its substrate [2-:'2P]Fru-2,6-P2 and the rateof dephosphoKC1 in solution A. The fraction containing the activity was rylation of ""P-phospho enzyme were similar to the rates of concentrated with Centricon-30 (Amicon) and subjected to rat liver enzyme (Fig. 7). The extent of phosphate incorpomolecular seive filtration on a Superose 12 column (Pharma- ration, however, was lower (0.42 mol of P incorporated per cia) which had been equilibrated with solution A. The purified mol subunit) than that (0.55 mol of P per mol) of the liver enzyme was concentrated with Centricon-30 again and stored Fru-2,6-bisphosphatase. T o confirm the existence of the same enzyme in rat testis frozen a t -80 "C. A typical example of this purification proin tissue exin uiuo, Fru-6-P,2-kinase:Fru-2,6-bisphosphatase cedure is summarized in Table I. The purified enzyme was homogeneous as judged by poly- tracts was phosphorylated with[2-'"P]Fru-2,6-P2 and subjected to polyacrylamide gel electrophoresis along with exacrylamide gel electrophoresis under denaturing conditions pressed phosphorylated enzyme. The results shown in Fig. 8 (Fig. 6). Physical Properties of the Expressed Enzyme-The ex- demonstrate thata '"P-labeled enzyme with identical M,was
15768
Testis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase
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 the2.0-kb mRNA specified the testis enzyme as determined by the testis enzyme DNA probe. ( b ) The expressed enzyme showedthe 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 aredifferent from other major isozymes of liver, skeletal muscle, and heart isozymes. However, some of the isozymes have been reported to havecomparableor higherkinase activity than the testis enzyme. The specific activities of those 0 1 2 3 preparations range from 100 to 180 milliunits/mg for beef TIME (min) heart (13, 39, 40) and pigeon muscle (41). However, some of FIG. 7. Phosphorylationanddephosphorylation of ex- these differences may be due to different assay conditions and pressed testis versus native liver Fru-2,6-bisphosphatase. In to different isozymic forms in the tissue. For example, we a final volume of 100 p1 the reaction mixture contained 0.5 PM Fru6-P,2-kinase:Fru-2,6-bisphosphatase, 100 mM Tris-HC1, pH 7.5, 2 have shown that the major isozyme (Mr 58,000) in bovine mM dithiothreitol, 10 mM MgC12, 0.2mM EDTA, and5 mM potassium heart has thespecific kinase activityof 61 milliunits/mg, but 54,000) shows 175 milliunits/mg. It is phosphate. The reaction was initiated with addition of 1 p M Fru-2,6- the minor isozyme (M, [2-:”P]P2 (1500 cpm/pmol), and incubated a t 30 “C. At various time interesting that the testisenzyme has the highest Fru-6-P,2intervals, 2 0 4 aliquots were removed and mixed with 180ml of cold kinase activity and the lowest Fru-2,6-bisphosphatase activ0.1 N NaOH containing 0.1 mg/ml bovine serum albumin. Aliquots ity. Since the Fru-2,6-P2 level in cells is controlled by the (40 ml) of alkali-treated samples were pipetted into 0.5 ml of cold relative activities of kinase/phosphatase, the high ratio of 10% trichloroaceticacid containing 0.1% bovine serumalbumin, filtered on a 0.45-pm Millipore filter (HA), andwashed with 15 ml of these two activities in testis may reflect the importance of cold trichloroacetic acid. The filters were counted in liquid scintilla- Fru-2,6-P2 ina glycolytic tissue such as testis. tion fluid. One of the important regulatory mechanisms of liver and is phosphoryheart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase lation and dephosphorylation discussed as in the introduction. 1 2 I t is possible that the testis enzyme also is regulated by the same mechanism. The testisenzyme was not phosphorylated 92.5Kby CAMP-dependentprotein kinase, unlikethe liver and heart 68Kisozymes, but similar to rat skeletal muscle enzyme. However, it was phosphorylated by protein kinaseC, and in this respect m the enzymewas similar to the bovine heart isozyme. The 45Ksignificance of the phosphorylationof the testisenzyme is not certain. The testis enzyme contained three potential phosphorylation sitesfor protein kinase C, namely Ser’, Ser28,and FIG.8. Phosphorylation of pure expressedrat testis Fru-6- Thr4“. Because of the opposite effectson theenzyme activities P,2-kinase:Fru-2,6-bisphosphataseand testis extract enzymedepending upon the location of the phosphorylation site(s), of great interest by Fru-2,6-P2. The phosphorylation by Fru-2,6-[2-”’P]P2 was per- i.e. N or C terminus of the enzyme (19), it is formed (for 30 s) as described for Fig. 7. Testis extract was prepared to distinguish these sites in the testis enzyme. Currently we as described under “Methods.” Lane I , the expressed enzyme; lane 2, are ascertaining thelocation of the phosphorylation site and testis extract. determining its effect on the activities of the bifunctional enzyme. These studies may elucidate the regulatory mechadetectable in testis extract, suggesting that the enzyme is nism of phosphofructokinase andglycolysis in testistissue. present in uiuo. A comparison of the aminoacid sequence of rat testisFruPhosphorylation by Protein Kinase C-The rat testis Fru- 6-P,2-kinase:Fru-2,6-bisphosphatase with those of rat liver 6-P,2-kinase:Fru-2,6-bisphosphatasecould not be phosphor- (42,43), rat heart (this paper), and bovine heart (22) enzymes ylated by CAMP-dependent protein kinase, unlike the rat is presented in Fig. 9. T o maximize the alignment of these liver or bovine heart isozymes. However, the testis enzyme sequences one amino acid gap had to be added to the testis was phosphorylated by protein kinase C and thephosphoryl- and two gapsto theliver enzymesequences. During thecourse ation required the presence of phosphatidyl serine, Ca2+,and of this work, we have also isolated from the same library a diolein (data not shown). The phosphorylation site was not cDNAclonewhichencodedfor ratheartFru-6-P,2-kidetermined, but based on a computer search for a potential nase:Fru-2,6-bisphosphatase.The deduced amino acidsesite for protein kinase C, the likely target serinewas localized quence of this clone corresponded to Pro’* to the C terminus in serine 28 or threonine443. of the sequence of the bovine heart enzyme, and those sequences of the heart enzymes were 94% identical. The rat DISCUSSION testis enzyme showed74,67, and64% similarity with rat liver, The cDNA clone for rat testis Fru-6-P,2-kinase:Fru-2,6-rat heart, andbovine heart enzymes, respectively. The major bisphosphatase reported hereinencoded a n enzymatically ac- differences amongthese sequences were in the N and C tive form of the enzyme. Our previous results, based on ion- termini where the regulatoryphosphorylation sites are 10exchange chromatography and immunoreaction,showed the cated.Although the C termini of the testis and the liver existence of a heart-type isozyme and an unidentified form of the isozyme which is more abundant in rat testis. The present ’R. Sakakibara and K. Uyeda, unpublished results.
Testis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase Testis Liver Heart (8) Heart ( R )
- ASPRELTQNPLKKIWHPYSNGRPAUL&SQR -- S R ESGNPASSSEQNNNSYETlUSLRISEIMCSW PlGELMTRIqKIWIPHSSSSSV~~G~S -
-Testis -Liver Heart (B) Heart (R) Testis -G Liver -G Heart (8) - S Heart ( R ) - A
Testis Liver Heart (B) Heart (R)
Heart (8) Heart (R)
-100
-
Testis Liver Heart (B) Heart ( R )
-- ~ -
Heart (B) Heart Liver (R)
-- FALR
Heart ( R )
-50
15769
residues, Lys117and Lys3"j, of the liver enzyme are also conserved in both the testis and heartenzymes. 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, thesubstrate, 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-6P,2-kinase do not react with Fru-2,6-bisphosphatasesites, which may suggest that theactive site is buried. The only site identified thus far is the active site, His'". The phosphoHis258as thereaction intermediate of Fru-2,6-bisphosphatase (38) and itsneighboring 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 sitesof Fru-B-P,2-kinase need to be elucidated. Acknowledgments-We wish to thank Nami Lee and Dawn Hancock for expert technical assistance and Dr. Sarah McIntire for her We also thank Dr. Melanie H. advice inmanuscriptpreparation. Cobb (University of Texas Southwestern Medical Center at Dallas, Department of Pharmacology) for protein kinase C.
O ~ T I O T ~ E O W ~ E I D A G V C E E M T Y F B I d D ~
E-350
A
REFERENCES
1. Uyeda, K., Furuya, E., Richards, C. S., and Yokoyama, M. (1982) 450 Mol. Cell Biochem. 48,97-120 2. Furuya, E., and Uyeda, K. (1981)J. Biol. Chem. 256, 7109-7112 3. El-Maghrabi, M. R., Claus, T. H., Pilkis, J., and Pilkis, S. J. -500 (1981)Biochem. Biophys. Res. Commun. 101, 1071-1077 LSSSN~IRRPRNYSVGSRPIP~P~ LSSSNTI~PRNYSVGSRPLKPLSPLRA 4. Van Schaftingen, E., and Hers, H. G. (1981)Biochem. Biophys. Res. Commun. 101,1078-1084 Heart (e) - LDTQEGADQPKTQAETSIRLPSPAPFTSPS -550 Heart ( R ) - LDMQEGADQPKTQVSIPVV 5. Van Schaftingen, E., Davies, D. R., and Hers, H. G. (1982)Eur. J. Biochem. 124, 143-149 6. Furuya, E., Yokoyama, M., and Uyeda, K. (1982)Biochem. BioFIG. 9. Comparison of amino acid sequences (in one-letter phys. Res. Commun. 105,264-270 code) of rat testis(testis),rat liver(liver),and bovine ( B )and rat (R)heart (heart) Fru-6-P,2-kinase:Fru-2,6-bisphospha- 7. El-Maghrabi, M. R., Claus, T . H., Pilkis, J., Fox, E., and Pilkis, S. J. (1982)J Biol. Chem. 257, 7603-7607 tase. The single amino acid gap was introduced in the testis and the 8. Rider, M. H., Foret, D., and Hue, L. (1985)Biochem. J. 231, liversequences to allow maximumsimilarity.Sequencepositions 193-196 whichallfour seenclosed in bores represent the amino acids in 9. Sakakibara, R., Kitajima, S., and Uyeda, K. (1984)J. Bid. Chem. quences are identical. The phosphorylation sites of liver and heart 259,41-46 enzymes are underlined. 10. El-Maghrabi, M. R., Pate, T. M., Murray, K. J., and Pilkis, S. J. (1984)J . Biol. Chem. 259, 13096-13103 enzymes are similar (13 out of 18 amino acids from Val4" to 11. van Schaftingen, E., and Hers, H. G . (1986)Eur. J. Biochem. the C terminus of the testis enzyme), their N-terminal se159,359-365 quences are different. On the other hand, both terminiof the 12. Kitamura, K., Uyeda, K., Kangawa, K., and Matsuo, H. (1989) J. Biol. Chem. 264,9799-9806 heart isozymes are completely different from those of the testis and the liver isozymes. It is noteworthy that the C- 13. Kitamura, K., and Uyeda, K. (1988)J Biol. Chem. 263, 90279033 terminal residues of bovine and rat heartenzymes containing 14. Taniyama, M., Kitamura, K., Thomas, H., Lawson, J. W . R., and the phosphorylation sites for CAMP-dependent protein kinase Uyeda, K. (1988)Biochem. Biophys. Res. Commun. 157, 949and protein kinase C (22) are nearly identical. Beyond these 954 terminal regions there are considerable numbers of amino 15. Furuya, E., Yokoyama, M., and Uyeda, K. (1982)Proc. Natl. Acad. Sci. U. S. A. 79,325-329 acid sequences that arehighly conserved (indicated inblocks). It is interesting that the C-terminal halves of these enzyme 16. El-Maghrabi,M. R.,Claus, T. H.,Pilkis, J., and Pilkis, S. J. Proc. Natl. Acad. Sci. U. S. A . 79,315-319 molecules (Fru-2,6-bisphosphatase domain) beginning from 17. van(1982) Schaftingen, E., and Hers, H. G. (1981)Biochem. Biophys. Leu'" of the testis enzyme are strikingly similar. Moreover, Res. Commun. 103,362-368 most of the differing amino acids are conservative substitu- 18. Richards, C. S., Yokoyama, M., Furuya, E., and Uyeda, K. (1982) tions. Among the highly conserved sequences, it is not surBiochem. Biophys. Res. Commun. 104,1073-1079 19. Kitamura, K., and Uyeda, K. (1987)J. Bid. Chem. 262,679-681 prising to find various substrate and product binding sites. The characteristic nucleotide binding site (44) is Gly-Leu- 20. Kitamura, K., Kangawa, K., Matsuo, H., and Uyeda, K. (1988) J. Biol. Chem. 263,16796-16801 Pro-Ala-Arg-Gly-Lys-Thr (residues 45-52of the testis en21. Narabayashi, H., Lawson, J. W . R., and Uyeda, K. (1985)J . Biol. zyme sequence) in these enzymes. The Fru-6-Pbinding sites, Chem. 260,9750-9758 as determined by affinity labeling experiments using N-bro- 22. Sakata, J., and Uyeda, K. (1990)Proc. Natl. Acad. Sci. U. S. A. moacetylethanolamine-P, are Cys'07 of the liver enzyme and 87,4951-4955 23. Richards, C. S., Yokoyama, M., Furuya, E., and Uyeda, K. (1982) the corresponding residues, Cys'05 and Cyslg6,of the heart Biochem. Biophys. Res. Commun. 104, 1073-1079 enzymes (45). The amino acid sequences surrounding these 24. Murray, K. J., El-Maghrabi, M. R., Kountz, P. D., Sukas, T. J., Cys residues are well conserved. We also described earlier Soderling, T. R., and Pilkis, S. J. (1984)J. Biol. Chem. 259, (37) conditions for the modification of the liver enzyme with 7673-7681 pyridoxal-P which results inselective modification of two Lys 25. Algaier, J . , and Uyeda, K. (1988)Biochem. Biophys. Res. Comresidues involved in Fru-2,6-P2 and ATP binding. These Lys mun. 153,328-333
Testis Fru-6-P,2-kinase:Fru-2,6-bisphosphatase 26. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13 27. Dotto, G . P., Horiuchi, K., and Zinder, N. D. (1984) J. Mol. Bid. 172,507-521 28. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Nutl. Acad. Sci. U. S. A. 74,5463-5467 29. Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977) Biochemistry 16,4743-4750 30. Saiki, R. K., Gelfand, D.H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239,487-491 31. Tabor, S., and Richardson, C. (1985) Proc. Nutl. Acud. Sci. U. S. A. 82,1074-1078 32. Studier, F. W., and Moffat, B. A. (1986) J . Mol. Biol. 189, 113130 33. Uyeda, K., Furuya, E., and Luby, L. J. (1981)J. Biol. Chem. 256, 8394-8399 34. Kitajima, S., Sakakibara, R., and Uyeda, K. (1984)J. Biol. Chem. 259,6896-6903 35. Laemmli, U. K. (1970) Nature 227, 680-685
36. Bradford, M. M. (1976)Anal. Biochem. 72, 248-254 37. Kitajima, S., Thomas, H., and Uyeda, K. (1985) J. Bwl. Chem. 266, 13995-14001 38. Stewart, H. B., El-Maghrabi, M.R., and Pilkis, S. J. (1985) J. Bid. Chem. 2 6 0 , 12935-12941 39. Rider, M. H., Foret, D., and Hue, L. (1985) Biochem. J. 231, 193-196 40. Rider, M. H., and Hue, L. (1986) Biochem. J. 240, 57-61 41. Van Schaftingen, E., and Hers, H. G. (1986) Eur. J. Biochem. 159,359-365 42. Darville, M. I., Crepin, K. M., Vandekerckhoue, J., Van Damme, J., Octave, J. N., Rider, M. H., Marchand, M. J., Hue, L., and Rousseau, G. G. (1987) FEBS Lett. 224, 317-321 43. Lively,M. O., El-Maghrabi, M.R., Pilkis, J., D’Angelo,G., Colosia, A. D., Ciavola, J.-A., Fraser, B. A., and Pilkis, S. J. (1988) J. Biol. Chem. 263,839-849 44. Rossman, M. G., Liljas, A., Branden, C. I., and Banaszak, L. J. (1975) in The Enzymes (Boyer, P., ed) 3rd Ed., vol. 11, pp. 61102, AcademicPress, New York 45. Kitamura, K., Uyeda, K., Hartman, F. C., Kangawa, K., and Matsuo, H. (1989) J. Biol. Chem. 264,6344-6348
