©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Isolation and Characterization of cDNA Encoding Human and Rat Brain Inositol Polyphosphate 4-Phosphatase (*)

F. Anderson Norris , Vorachart Auethavekiat , Philip W. Majerus

From the (1)Division of Hematology-Oncology, Departments of Internal Medicine and Biological Chemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Inositol polyphosphate 4-phosphatase, an enzyme of the inositol phosphate signaling pathway, catalyzes the hydrolysis of the 4-position phosphate of inositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate, and phosphatidylinositol 3,4-bisphosphate. The amino acid sequences of tryptic and CNBr peptides of the enzyme isolated from rat brain were determined. Degenerate oligonucleotide primers based on this sequence were used to amplify a 74-base pair polymerase chain reaction product. This product was used to isolate a 5607-base pair composite cDNA, which had an open reading frame encoding a protein with 939 amino acids with a predicted molecular mass of 105,588 Da. The rat brain polymerase chain reaction product was used as a probe to isolate a human brain cDNA that predicts a protein with 938 amino acids and a molecular mass of 105,710 Da. Remarkably, the human and rat proteins were 97% identical. Recombinant rat protein expressed in Escherichia coli catalyzed the hydrolysis of all three substrates of the 4-phosphatase. Northern blot hybridization indicates that the 4-phosphatase is widely expressed in rat tissues with the highest levels of expression occurring in brain, heart, and skeletal muscle. Polyclonal antiserum directed against the carboxyl terminus of the 4-phosphatase immunoprecipitated >95% of the 4-phosphatase activity in crude homogenates of rat brain, heart, skeletal muscle, and spleen, suggesting that this enzyme accounts for the 4-phosphate activ-ity present in rat tissues. This antiserum also immunoprecipitated the 4-phosphatase from human platelet sonicates.


INTRODUCTION

Inositol polyphosphate 4-phosphatase is a Mg-independent enzyme that catalyzes the hydrolysis of the 4-position phosphate of inositol 3,4-bisphosphate (Ins(3,4)P)()and inositol 1,3,4-trisphosphate (Ins(1,3,4)P)(1) . Recently, the 4-phosphatase was shown to catalyze the hydrolysis the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P) with a first order rate constant 120- and 900-fold greater than that of inositol 3,4-bisphosphate and inositol 1,3,4-trisphosphate, respectively(2) . This suggests that the 4-phosphatase is an enzyme involved in the D-3 phosphate containing phosphatidylinositol pathway.

Phosphatidylinositols phosphorylated at the D-3 position are formed in response to extracellular agonists. The D-3 phosphate-containing lipids, PtdIns(3)P, PtdIns(3,4)P, and PtdIns(3,4,5)P, are believed to be formed chiefly by the PtdIns-3 kinase that has been shown to physically associate with a variety of activated tyrosine kinases(3, 4) . Activation of the PtdIns 3-kinase has been implicated in the signal transduction of platelet-derived growth factor receptor-mediated mitogenesis(5) , the oxidative burst of neutrophils (6), and translocation of the glucose transporter to the plasma membrane(7) . The D-3 phosphate-containing phosphatidylinositols are not hydrolyzed by phospholipase C enzymes (8, 9) and thus may act directly as signaling molecules in these processes. The targets for these potential second messengers are yet to be defined, although PtdIns(3,4)P and PtdIns(3,4,5)P have recently been shown to activate isoenzymes of protein kinase C in vitro(10, 11) .

We now report the isolation of cDNAs encoding rat and human brain 4-phosphatases that are 97% identical at the amino acid level. We demonstrate the expression of active recombinant enzyme in Escherichia coli and have determined the tissue distribution of expression by Northern blot analysis. In addition, an antibody directed against the 4-phosphatase is shown to immunoprecipitate >95% of the observed 4-phosphatase activity from the supernatant of crude extracts of several rat tissues and human platelets.


EXPERIMENTAL PROCEDURES

Materials

Frozen unstripped rat brains were purchased from Pel-Freeze. Unlabeled Ins(1,3,4)P and leupeptin were purchased from Calbiochem. [H]Ins(3,4)P, unlabeled Ins(3,4)P, and [P]PtdIns(3,4)P were prepared as described(2) . GeneAmp dNTP, 10 PCR buffer, and AmpliTaq DNA polymerase were purchased from Perkin-Elmer Corp. Oligonucleotides were synthesized by the Washington University Protein and Nucleic Acid Laboratory. The Colony/Plaque Screen hybridization filters, Aquassure scintillation mixture, [P]dATP, [P]ATP, and [H]Ins(1,3,4)P were purchased from DuPont NEN. The sequencing grade trypsin (modified), T4 polynucleotide kinase, and the random primed labeling kit were purchased from Boehringer Mannheim. phenylmethylsulfonyl fluoride, keyhole limpet hemocyanin, bromoacetic acid N-hydroxysuccinimide ester, protein A-Sepharose, and CNBr were from Sigma. A Uni-ZAP rat brain cDNA library (oligo(dT) primed), a Zap II human fetal brain cDNA library (oligo(dT) and random primed), and E. coli strain XL1-Blue were purchased from Stratagene. A rat brain 5`-STRETCH library (random primed), rat brain QUICK-clone cDNA, and rat brain multi-tissue Northern blot were purchased from Clontech. The Sequenase (version 2.0) DNA sequencing kit was purchased from U. S. Biochemical Corp. [thio-S]dATP was purchased from Amersham Corp. The Wizard miniprep kit was purchased from Promega. The LongRanger gel solution was from Fisher. The TA Cloning kit was from Invitrogen. The QIAexpress PQE-32 expression vector and Ni-NTA agarose were purchased from Qiagen.

Protein Sequencing

The 4-phosphatase was partially purified from rat brain as previously described(2) . The purified protein was run on a 6% preparative SDS-polyacrylamide gel electrophoresis gel, and the 110-kDa protein (35 µg) band was excised from the gel and electroeluted, acetone precipitated, and then either treated with trypsin or CNBr; the resulting peptides were then separated by high pressure liquid chromatography and sequenced as previously described(12) .

cDNA Cloning

The degenerate oligonucleotides 5`-GAATTCCCNGTNCTNTTYAAYGT-3` (sense) and 5`-GAGCTCTTRTCNCCRAANCKYTC-3` (antisense) were derived from the amino acid sequence of a CNBr peptide RVQPVLFNVGINEQQTLAERFGDTSLQ (Fig. 2, amino acids 744-770) with the addition of a 5`-EcoRI and 5`-XhoI restriction site to the sense and antisense oligonucleotides, respectively. These oligonucleotides (350 ng each) were used as primers in a 20-µl PCR containing 0.6 units of AmpliTaq DNA polymerase, 0.25 mM GeneAmp dNTP, 1 PCR buffer, and 1 ng of rat brain QUICK-clone cDNA (Clontech). The reaction was carried out for 30 cycles with a 1-min denaturation step at 94 °C, a 1.5-min annealing step at 50 °C, and a 2-min extension step at 72 °C. PCR amplification was then repeated using 1 µl of the first reaction as template using the same primers and cycling program. The resulting 74-bp PCR product (PCR product A) was subcloned using a TA Cloning kit (version 1.3) (Invitrogen) and sequenced to verify that the PCR product predicted the correct amino acid sequence of the CNBr peptide. The product was end labeled with [P]ATP using T4 polynucleotide kinase (Boehringer Mannheim) and used to probe a rat brain Uni-ZAP cDNA library (Stratagene). Clones were plaque purified and rescued into Bluescript SK± plasmid using the R408 helper phage following the manufacturer's protocol (Stratagene). One of the clones isolated, clone 4, was used as template to generate PCR product B. The primers 5`-AGTCTGTGAGTTGGAAGAGT-3` (sense) and 5`-GAGCCGTTCTGCATAGTAGC-3` (antisense) derived from the 5`-sequence of clone 4 were used for PCR under the conditions described above. The resulting 480-bp product was labeled with P using a random primed labeling kit (Boehringer Mannheim) and then used to screen the Uni-ZAP cDNA library (Stratagene) and the random primed rat brain 5`-STRETCH cDNA library (Clontech). cDNA libraries were screened by probing plaques bound to duplicate Colony/Plaque Screen hybridization filters (Dupont NEN) following the manufacturer's protocol for aqueous hybridization. DNA sequencing reactions were performed using the Sequenase (version 2) kit (U. S. Biochemical Corp.) except for a short C-rich segment of the 3`-UTR (bp 3480-3540), which was sequenced with Taq polymerase using the manufacturer's protocol (Perkin Elmer). Both strands of the open reading frame were sequenced. The human cDNA was isolated by the same protocol from the fetal brain cDNA library mentioned above using P end-labeled PCR product A as probe.


Figure 2: Nucleotide and predicted amino acid sequence of the rat 4-phosphatase cDNA. The numbering of the nucleotides (leftside) and the amino acids (rightside) begin at the start methionine. Peptide sequence obtained from purified 4-phosphatase are underlined; solidlines indicate tryptic peptides, and dashedlines indicate CNBr peptides. ATTTA sequences implicated in mRNA instability are also underlined. The polyadenylation signal is boxed. There is also a box at amino acid 574 indicating the 11 amino acid deletion found in clone 13.



Bacterial Expression of Inositol Polyphosphate 4-Phosphatase

The 689-bp AvaI-BamHI fragment of clone 6b and the 2678-bp BamHI-HindIII fragment of clone 4 (Fig. 1) were subcloned into Bluescript SK± cut with AvaI and HindIII. The 3379-bp KpnI-HindIII fragment of this construct was then subcloned into the PQE-32 expression vector (Qiagen). The resulting construct expressed an amino-terminal six histidine tag and the 4-phosphatase minus amino acid residues 1-13. The recombinant protein was expressed in XL1-blue strain of E. coli (Stratagene). Bacteria were grown at 37 °C in LB broth containing 100 µg of ampicillin/ml until an A of 0.6 OD was reached. Isopropyl-1-thio--D-galactopyranoside (1 mM) was then added, and culture growth was continued for 3 h at room temperature. Bacteria were harvested by centrifugation at 4000 g for 20 min, and the pellet was resuspended in a buffer containing 50 mM potassium phosphate, 10 mM Tris, pH 8.0 (buffer A), with 1 µg of lysozyme/ml (2 ml/g, wet weight) and incubated on ice for 30 min. The suspension was then sonicated on ice using a probe sonicator at 200 watts for 30-s intervals until the cells were lysed (approximately 2 min). The lysate was centrifuged at 20,000 g for 20 min. The supernatant was then mixed with Ni-NTA-agarose (1 ml of 50% slurry/ml of supernatant) for 30 min at 4 °C. The Ni-NTA-agarose was transferred to a 1-cm diameter column and washed with five column volumes of buffer A. The column was then washed with four column volumes of buffer A containing 20 mM imidazole, pH 8.0, and four column volumes of buffer A containing 250 mM imidazole, pH 8.0. Fractions containing 4-phosphatase activity were pooled and concentrated using a centriprep 30 spin concentrator (Amicon).


Figure 1: Independent clones isolated from cDNA libraries. The relationship between the composite 4-phosphatase clone and the isolated cDNA clones and probes used for screening are shown. The thinbars indicate the location of the sequence of each independent cDNA clone: clones 4, 7, 13, and 15 (isolated from a Stratagene library), clones 2B, 6B, and 10A (isolated from Clontech library), and human clones 12A and 12C (isolated from a Stratagene library). A partial restriction map of the composite cDNA is given for ApaI (A), BamHI (B), EcoRI (E), and HindIII (H). The relative location of PCR products A and B are also indicated. Note that clone 13 is missing 11 amino acids (at 574) present in other rat and human clones.



Northern Blot Analysis

PCR product B was P-labeled using the random hexamer procedure described above. A rat multi-tissue Northern blot was then probed following the protocol recommended by the manufacturer (Clontech). The blot was stripped of probe by boiling in 1% SDS for 10 min and reprobed with P-labeled -actin control probe.

Production of Rabbit Polyclonal Peptide Antiserum

The peptide CPPEGTYGKVET corresponding to the carboxyl-terminal tryptic peptide was synthesized by Washington University Protein and Nucleic acid Laboratory and coupled to keyhole limpet hemocyanin with bromoacetic acid N-hydroxysuccinimide ester(13) . Rabbit polyclonal serum was raised to antigen by Pocono Rabbit Farms and Laboratory (Cacadensis, PA).

Immunoprecipitation of Inositol Polyphosphate 4-Phosphatase

Rat brain homogenate was prepared by homogenizing 10 minced unstripped rat brains in a polytron using 20 mM HEPES, pH 7.5, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg of leupeptin/ml (homogenization buffer). The supernatant was prepared by centrifuging the homogenate for 30 min at 20,000 g. Immunoprecipitations were performed by adding 5 µl of a mixture of preimmune rabbit serum and some quantity of immune serum (0-0.2 µl) to 100 µl of supernatant and incubating at 4 °C for 4 h. Then, 90 µl of a 50% slurry of a protein A-Sepharose CL-4B in homogenization buffer was added and mixed overnight at 4 °C. The amount of 4-phosphatase remaining in the supernatant was then assayed using both a soluble and lipid substrate as previously described(2) , except that 0.1% Triton X-100 was substituted for the octylglucoside in the lipid assay buffer.


RESULTS

Sequence and Structural Features of 4-Phosphatase cDNA

The amino acid sequence was determined for tryptic and CNBr peptides derived from rat brain inositol polyphosphate 4-phosphatase. A 74-base pair PCR product was obtained using degenerate sense and antisense oligonucleotides derived from the amino acid sequence of a CNBr peptide as described under ``Experimental Procedures.'' This PCR product was labeled and used to screen an oligo(dT) primed Stratagene Unizap rat brain library. Three overlapping clones obtained from this screen were sequenced (Fig. 1). A 480-base pair PCR product derived from the 5`-end of clone 4 was then used to screen both a Stratagene Unizap library and a Clontech random primed rat brain 5`-stretch library. One additional clone obtained from the Stratagene library and three clones from the Clontech library were sequenced. The composite cDNA contained an open reading frame that predicted a protein of 939 amino acids of 105,588 Da. This corresponds closely to the molecular mass of 110 kDa determined for the bovine 4-phosphatase by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography(1) . The open reading frame that contains an ATG that conforms to the Kozak consensus (14) and an in-frame stop TGA occurs at nucleotide -39 (Fig. 2). The predicted amino acid sequence of the open reading frame contains the sequences of seven tryptic and two CNBr peptides that were obtained from the native enzyme (Fig. 2). The 3`-UTR contains eight ATTTA sequences that correspond to the AUUUA motifs shown to be present in mRNAs that are rapidly degraded(15) . The 3`-UTR terminates in a poly(A) tail preceded by a AAUAAA polyadenylation signal (Fig. 2). One of the clones isolated (clone 13) lacked 33 nucleotides corresponding to amino acids 574-585 ( Fig. 1and Fig. 2). This may indicate an alternatively spliced form of 4-phosphatase. The PCR product A was used to isolate a cDNA encoding human inositol polyphosphate 4-phosphatase from a human fetal brain cDNA library. Two clones were isolated and sequenced, one of which (clone 12A) contained the entire open reading frame of human 4-phosphatase (Fig. 1). Remarkably, the human and rat proteins are 97% identical with most of the amino acid substitutions being conservative (Fig. 3). The rat 4-phosphatase is one amino acid longer than the human 4-phosphatase as a result of a serine inserted at position 489. The nucleotide sequences of the human and rat clones were 90% identical throughout the coding region but only 60% identical in the 5`-UTR.


Figure 3: Alignment of the amino acid sequences of rat and human 4-phosphatases. The amino acid sequence of rat (top) and human (bottom) were aligned using the Genetics Computer Group program GAP (23). Identical amino acids are indicated by verticallines. A doubledot indicates a conservative substitution, and a singledot indicates a change caused by a single nucleotide change.



Northern Blot Analysis

PCR product B was end labeled and hybridized to a multi-tissue Northern blot to determine the tissue distribution of expression (Fig. 4). The results indicate that 4-phosphatase is widely expressed in rat tissues, the relative expression level being brain > heart = skeletal muscle > spleen = kidney > lung = testis. The size of the 4-phosphatase mRNA transcript was estimated to be 6.2 kilobases, indicating that the composite cDNA clone lacks approximately 0.6 kilobases of 5`-UTR. The nature of the hybridizing 12-kilobase transcript found primarily in brain is unknown.


Figure 4: Northern blot of rat mRNA isolated from various tissues. Lanes1-8 are mRNA isolated, respectively, from heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testes screened with 4-phosphatase PCR B probe (upperpanel) or -actin control probe (lowerpanel).



Heterologous Expression of 4-Phosphatase

A construct that contains a histidine-tagged version of 4-phosphatase was expressed in E. coli. This construct contains 6 histidine residues at the amino terminus and lacks the first 13 amino acids of the predicted protein. Lysates of bacteria expressing this construct were found to contain 4-phosphatase activity, whereas extracts from bacteria-expressing vector only had no detectable activity (data not shown). The soluble activity was partially purified on a Ni-NTA affinity column as described under ``Experimen-tal Procedures'' and was shown to hydrolyze Ins(3,4)P, Ins(1,3,4)P, and PtdIns(3,4)P, the three substrates of 4-phosphatase (Fig. 5). There was no hydrolysis of Ins(1,4)P, an inositol phosphate that is not a substrate of the 4-phosphatase, by this preparation (data not shown). These experiments indicate that the recombinant enzyme has the same substrate specificity as the native enzyme. We estimated the concentration of recombinant 4-phosphatase in the partially purified extracts by comparing immunoblots of varying amounts of bacterial extract versus native 4-phosphatase. This estimation indicated that the recombinant protein was only 16% as active as the native enzyme (4 µmol of Ins(3,4)P hydrolyzed/min/mg of protein versus 25 µmol of Ins(3,4)P hydrolyzed/min/mg of protein). Whether this results from the addition of the histidine tag to the amino terminus, deletion of the first 13 amino acids, or inactivation during purification is unknown.


Figure 5: Time course of the hydrolysis of 4-phosphatase substrates by recombinant enzyme. 0.6 µg () or 1.2 () µg of partially purified histidine-tagged recombinant enzyme was incubated with (A) 10 µM Ins(3,4)P or 10 µM (B) Ins(1,3,4)P. C, TLC plate of PtdIns(3,4)P hydrolyzed by 0.6 µg of recombinant enzyme.



Immunoprecipitation of 4-Phosphatase

A polyclonal antiserum directed against a carboxyl-terminal peptide of the 4-phosphatase was capable of immunoprecipitating >95% enzyme activity present in crude rat brain supernatant (Fig. 6). The fraction of activity remaining in the supernatant after immunoprecipitation with increasing amounts of antiserum was identical for Ins(3,4)P and PtdIns(3,4)P, indicating that the same enzyme was responsible for catalyzing the hydrolysis of both substrates (Fig. 6). In addition, approximately 95% of the activity could be removed from supernatants of human platelets and rat heart, skeletal muscle, and spleen using this antiserum, indicating that this enzyme is the primary 4-phosphatase activity found these tissues (data not shown).


Figure 6: Immunoprecipitation of 4-phosphatase activity from crude rat brain supernatant. The activity of 4-phosphatase remaining in the supernatant after immunoprecipitation with increasing amounts of rabbit polyclonal antiserum directed against a COOH-terminal peptide was determined for the substrate Ins(3,4)P () or PtdIns(3,4)P ().




DISCUSSION

We have isolated cDNA molecules encoding human and rat brain 4-phosphatase. This conclusion is based on the following criteria: 1) the cDNA encodes a protein of the appropriate molecular weight that contains all of the peptide sequence obtained from the native enzyme, 2) bacterially expressed recombinant protein catalyzes the hydrolysis of the three substrates of 4-phosphatase, and 3) antisera raised against the carboxyl-terminal peptide immunoprecipitates >95% of the 4-phosphatase activity from supernatants of human platelets and rat brain, heart, skeletal muscle, and spleen. This suggests that this enzyme may be the only 4-phosphatase expressed in these tissues. In addition, Northern blot analysis indicates that the expression of mRNA in various rat tissues correlates with the enzyme activity levels previously reported for bovine tissues, with brain having the highest activity(16) .

The amino acid sequence predicted by the rat brain 4-phosphatase cDNA is 97% identical to that of human 4-phosphatase. The majority of differences between the two sequences were conservative substitutions except for deletion of one amino acid from the human sequence. A TBLASTN search (17) of the Genbank data base disclosed that the 4-phosphatase amino acid sequence is 25% identical over 300 amino acids to a predicted gene found in Caenorhabditis elegans cosmid R01H10, suggesting that it may encode a related protein. This remarkable conservation between species suggests that 4-phosphatase has an essential function. The amino acid sequence of 4-phosphatase has no significant similarity to any other inositol phosphate metabolizing enzyme that has been cloned to date. Most notably, inositol polyphosphate 1-phosphatase has no similarity to 4-phosphatase even though it hydrolyzes one of the same substrates (Ins(1,3,4)P(12) . However, the 4-phosphatase is the first Mg-independent inositol phosphate phosphatase to be cloned, and sequence similarity with other members of this category such as the inositol polyphosphate 3-phosphatase (18) and polyphosphoinositide phosphatase (19) may yet be observed.

Several features of the 4-phosphatase sequence suggest that its expression may be regulated. The 3`-UTR contains eight ATTTA motifs that have been shown to be responsible for rapid degradation of mRNA (15). The amino acid sequence of the 4-phosphatase contains two potential PEST sequences. PEST sequences are proline, glutamic acid, aspartic acid, serine, and threonine-rich domains that have been shown to be common in proteins that are rapidly degraded by the calcium-dependent thiol protease, calpain(20) . Potential PEST sequences were evaluated using the computer program PEST-FIND (21). PEST scores greater than 0 are considered strong PEST regions, and those between 0 and -5 are considered weak PEST regions(20) . The 4-phosphatase has two PEST domains. These are KASPTSTEEEQVMLR (Fig. 2, amino acids 486-500) and RLHGEGGEDVFPCSSTCSSK (Fig. 2, amino acids 555-574) with PEST scores of 2.1 and 0.5, respectively. Proteolysis by calpain may explain the instability of 4-phosphatase in the absence of EDTA(1) . 4-phosphatase also contains several potential protein kinase phosphorylation sites. Motifs for protein kinase C, multifunctional calmodulin kinase II, myosin-I heavy chain kinase, S6 kinase, proline-dependent kinase, and casein kinase I and II are all present(22) .

The cloning of the 4-phosphatase will allow its role in the inositol phosphate signaling pathway to be studied further. The recent observation that PtdIns(3,4)P is also a substrate for this enzyme (11) suggests that it may have a role in controlling the levels of this potential messenger. Expression of the 4-phosphatase in eukaryotic cells will allow us to study the role of the enzyme in metabolizing PtdIns(3,4)P and may prove to be a powerful tool for studying the signaling function of PtdIns(3,4)Pin vivo.


FOOTNOTES

*
This research was supported by Grants HL 14147, HL 16634 (Specialized Center for Research in Thrombosis), HL 16634, and Training Grant HL 07088. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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/EMBL Data Bank with accession number(s) U26397, U26398.

The abbreviations used are: Ins(3,4)P, inositol 3,4-bisphosphate; Ins(1,4)P, inositol 1,4-bisphosphate; Ins(1,3,4)P, inositol 1,3,4-trisphosphate; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(3,4)P, phosphatidylinositol 3,4-bisphosphate; PtdIns(3,4,5)P, phosphatidylinositol 3,4,5-trisphosphate; PCR, polymerase chain reaction; UTR, untranslated region; bp, base pair(s).


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