The cDNA cloning and characterization of inositol polyphosphate 4-phosphatase type II. Evidence for conserved alternative splicing in the 4-phosphatase family.

Inositol polyphosphate 4-phosphatase (4-phosphatase) is a Mg2+-independent enzyme that catalyzes the hydrolysis of the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 3,4-bisphosphate. We have isolated cDNA encoding a 105,257-Da protein that is 37% identical to the previously cloned 4-phosphatase. Recombinant protein was expressed in Escherichia coli and shown to hydrolyze all three 4-phosphatase substrates with enzymatic properties similar to the original enzyme. We designate the original 4-phosphatase and the new isozyme as inositol polyphosphate 4-phosphatase types I and II, respectively. 4-Phosphatase II is highly conserved with the human and rat enzymes having 90% amino acid identity. A conserved motif between 4-phosphatase I and II is the sequence CKSAKDRT that contains the Cys-Xaa5-Arg active site consensus sequence identified for other Mg2+-independent phosphatases. Northern blot analysis indicated that 4-phosphatase II is widely expressed with the highest levels occurring in the skeletal muscle and heart. In addition, cDNA encoding alternatively spliced forms of human 4-phosphatase I (107, 309 Da) and rat 4-phosphatase II (106,497 Da) were also isolated that encode proteins with a putative transmembrane domain near their C termini. These alternatively spliced forms were expressed as recombinant proteins in E. coli and SF9 insect cells and found to possess no detectable enzymatic activity suggesting that additional factors and/or processing may be required for these alternatively spliced isozymes.

In this study we report the cloning of a cDNA that encodes an isozyme of 4-phosphatase with 37% amino acid identity to the 4-phosphatase originally characterized. In addition, alternatively spliced cDNA of both isozymes of 4-phosphatase were identified that appear to encode C-terminal transmembrane domains. We have designated these 4-phosphatase isozymes as type I and type II with the hydrophilic and hydrophobic spliced forms as ␣ and ␤, respectively.  (3,4)P 2 were prepared as described previously (8). All restriction enzymes were purchased from Boehringer Mannheim.

Materials
Cloning of Type II 4-Phosphatase cDNA-A 74-base pair PCR product was amplified from rat brain Quick-clone cDNA (CLONTECH) using degenerate oligonucleotides that were based on the amino acid sequence obtained from the type I 4-phosphatase CNBr peptide RVQPVLFNVGINEQQTLAERFGDTSLQ. This PCR product was used * This research was supported by Grants HL 16634 and HL 55672 from the National Institutes of Health and the American Society of Hematology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
to screen a rat brain Uni-ZAP cDNA library (Stratagene) as described previously (9). During the process of cloning rat type I 4-phosphatase cDNA, clones were isolated that encoded 4-phosphatase type II. Fulllength rat 4-phosphatase II cDNA clones were isolated from the rat brain Uni-ZAP library (Stratagene) by hybridization screening using as probe a 413-base pair PCR product amplified from the 5Ј-end of a rat cDNA (clone 9) with the oligonucleotides 5-AGAACACGGCCAAG-GCAAAG-3Ј (sense) and 5Ј-GTTTGTCAACTTTAGCGATA-3Ј (antisense) using PCR conditions described previously (9) with the exception that 200 ng of each oligonucleotide was used. Human 4-phosphatase II EST clones, yy10a10, yx97 h10, yx36 g09, and zm11f05 were identified by a BLAST search (13) of the GenBank data base and were obtained from the American Type Culture Collection. Additional human cDNA clones were isolated from a human brain Lambda ZAP II cDNA library (Stratagene) by hybridization screening using a 362-base pair PCR product amplified from the EST clone yy10a10 using the oligonucleotides 5Ј-GCAGGTACACAGCCCTCACT-3Ј(sense) and 5Ј-AGAGCA-CTCCTGACAAGTTG-3Ј (antisense) under the same PCR conditions used to generate the 413-base pair rat 4-phosphatase PCR product. The human type I␤ cDNA clone was isolated from the Lambda Zap II human fetal brain cDNA library (Stratagene) using the 480-base pair PCR product used in the original cloning of type I␣ 4-phosphatase cDNA (9).
All DNA probes were 32 P-labeled using a random primed labeling kit (Boehringer Mannheim) using the manufacturer's protocol.
DNA Sequencing-The rat 4-phosphatase II clones were sequenced using the Sequenase kit version 2.0 (U. S. Biochemical Corp.) as described previously (9). The human 4-phosphatase II clones were sequenced using ABI Prism dye terminator cycle sequencing kit (Perkin Elmer) using the manufacturer's protocol with sequence reactions analyzed by the Nucleotide Sequencing Core Lab at Washington University School of Medicine.
Northern Analysis-The human 4-phosphatase II PCR product was 32 P-labeled using a random primed labeling kit (Boehringer Mannheim) and used to probe a human multi-tissue Northern blot (CLONTECH) following the manufacturer's protocol.
Bacterial Expression of Recombinant 4-Phosphatases-The expression construct for 6-histidine-tagged rat 4-phosphatase II was prepared by ligating the 2898-base pair XhoI-KpnI fragment of a rat 4-phosphatase II clone into the PQE-31 bacterial expression vector (Qiagen) that was cut with KpnI and SalI. The expression construct for 6-histidinetagged human 4-phosphatase I was prepared by ligating the 2928-base pair XhoI-EcoRI fragment of a human 4-phosphatase I clone into pTrcAHis bacterial expression vector (Invitrogen) that was cut with XhoI and EcoRI. Bacteria containing these expression vectors were grown at 37°C in 1 liter of LB broth until an A 600 of 0.6 optical density was reached. Isopropyl-1-thio-␤-D-galactopyranoside (1 mM) was added. After 5 h at room temperature, cells were harvested, and the protein was purified on Ni-nitrilotriacetic acid-agarose as described previously (9). The protein was dialyzed and concentrated in a ProDiCon concentrator (Spectrum) containing 20 mM Hepes, 2 mM EDTA, 100 mM NaCl, 5 mM dithiothreitol, and 0.1% octylglucoside (pH 7.5) and frozen at Ϫ140°C prior to enzyme characterization. The amount of recombinant enzyme in these preparations was determined by Coomassie Blue staining of protein separated by polyacrylamide gel electrophoresis compared with molecular weight standards. The recombinant enzyme in these preparations was only partially purified being 50 -60% of the total protein. The enzymatic properties of the partially purified 4-phosphatase were readily characterized because control bacterial lysates contain no detectable phosphatase activity under the assay conditions.
Miscellaneous Techniques-The 4-phosphatase soluble and lipid substrate assays were performed as described previously (9). Protein concentration was determined using the Bio-Rad protein assay. Enzyme kinetic data were analyzed using Kcat 1.3 software (Biometallics, Inc.). The amino acid sequence homology was analyzed using DNASTAR Megalign software (DNASTAR, Inc.)

RESULTS AND DISCUSSION
During the original cloning of 4-phosphatase I cDNA, we initially obtained a 74-base pair PCR product using degenerate primers derived from the amino acid sequence of a CNBr pep-tide from a purified rat brain 4-phosphatase (9). The PCR product predicted the amino acids of the CNBr peptide suggesting that it was amplified from 4-phosphatase cDNA. However, analysis of several PCR products indicated the presence of two PCR products that differed at nucleotides in the wobble positions of four codons. This sequence variation suggested the existence of another cDNA encoding a protein related to the 4-phosphatase, and further cDNA library screening yielded clones that encode an enzyme that we have designated inositol polyphosphate 4-phosphatase II (4-phosphatase II).
The 4-phosphatase II composite cDNA sequence consists of 3178 base pairs with an open reading frame predicting a protein with a molecular mass of 105,257 Da (Fig. 1). The open reading frame begins at a consensus start methionine (14) with an upstream stop codon in the Ϫ29 position. The 3Ј-end of this cDNA terminates 54 base pairs after the stop codon without a poly(A) tail suggesting that additional 3Ј-untranslated region remains to be identified. The human 4-phosphatase II cDNA sequence was also determined from the composite sequence of four cDNA clones isolated from a human brain cDNA library and four human EST clones. The human 4-phosphatase II open reading frame predicts a protein with a molecular mass of 104,744 Da. As shown in Fig. 2, the rat and human type II 4-phosphatases are highly conserved with 90% identical amino acid residues for the entire length of the proteins. Unlike 4-phosphatase I, the human and rat 4-phosphatase II enzymes do not possess PEST sequences; proline, aspartic acid/glutamic acid, serine/threonine rich motifs common in substrates of the calcium-dependent protease, calpain (15).
The type I and type II rat 4-phosphatases are 37% identical at the amino acid level (Fig. 3). In some regions the similarity is much greater, for example the C-terminal antipeptide antibody against 4-phosphatase I described previously (9) also precipitates type II enzyme (data not shown). The motif CKSAK-DRT is conserved between the type I and type II 4-phosphatases and contains the active site consensus sequence Cys-Xaa 5 -Arg identified in several other Mg 2ϩ -independent phosphatases including protein tyrosine phosphatases, dual specificity protein phosphatases, and low molecular weight acid phosphatases (16). The conserved cysteine within the Cys-Xaa 5 -Arg motif functions as the nucleophile for catalysis, and cysteine modifying reagents are potent inhibitors of this class of phosphatases (17). Both type I and type II rat 4-phosphatases are completely inactivated by the cysteine modifying reagent N-ethylmaleimide, a result consistent with these enzymes being Cys-Xaa 5 -Arg phosphatases (data not shown). Additional studies using site-directed mutagenesis will evaluate the role of the CKSAKDRT motif in 4-phosphatase catalysis.
The enzymatic properties of recombinant rat 4-phosphatase II are similar to those reported for human 4-phosphatase I. As shown in Fig. 4A, 4-phosphatase II catalyzes the hydrolysis of PtdIns(3,4)P 2 to phosphatidylinositol 3-phosphate in a timeand enzyme concentration-dependent manner. The first order rate constant for PtdIns(3,4)P 2 hydrolysis using 0.6 ng of either recombinant rat 4-phosphatase II or human 4-phosphatase I was 0.45 ϫ 10 Ϫ2 sec Ϫ1 and 1.1 ϫ 10 Ϫ2 sec Ϫ1 , respectively. The Lineweaver-Burk analysis of 4-phosphatase II hydrolysis of the soluble inositol phosphate substrates indicates a K m for Ins(3,4)P 2 and Ins(1,3,4)P 3 of 39 and 34 M, respectively and a V max for Ins(3,4)P 2 and Ins(1,3,4)P 3 of 26 and 22 mol/min/mg enzyme, respectively (Fig. 4B). These K m and V max values are similar to those measured for recombinant human 4-phosphatase type I hydrolysis of Ins(3,4)P 2 (K m ϭ 28 M; V max ϭ 32 mol/min/mg) and Ins(1,3,4)P 3 (K m ϭ 46; V max ϭ 28 mol/min/ mg). Other properties similar to those reported for 4-phosphatase I are that 4-phosphatase II activity is optimal between pH 7 and pH 8 and is strongly inhibited by inositol hexakisphosphate with an IC 50 of 50 M (data not shown) (7,8).
Northern blot analysis of the expression of human 4-phosphatase II is shown in Fig. 5. Four primary transcripts of 9.5, 5.3, 3.9, and 2.9 kilobases are detected. The 5.3-kilobase transcript is detected only in the brain. The relative abundance of the other transcripts is skeletal muscle Ͼ heart Ͼ Ͼ brain ϭ placenta ϭ pancreas Ͼ liver Ͼ kidney Ͼ lung. The pattern of 4-phosphatase II expression contrasts with that of 4-phosphatase I, which has the highest level of expression in the brain (9) suggesting that type I and type II isozymes may have tissuespecific functions.
Alternately spliced forms of human type I and rat type II 4-phosphatase cDNA clones were isolated that predict proteins of 107,309 Da and 106,497 Da, respectively. These splice variants encode hydrophobic C termini that contain putative membrane-spanning domains (Fig. 6). The splice junctions for these alternatively spliced forms of types I and II 4-phosphatase occur 38 codons 3Ј of the potential active site motif following the conserved amino acids CMR (Fig. 6). This alternative splic-ing of 4-phosphatase II mRNA may explain in part the presence of the multiple sized transcripts in the 4-phosphatase II Northern blot. The alternatively spliced forms of types I and II 4-phosphatase expressed in bacteria or SF9 insect cells possess no detectable 4-phosphatase activity for PtdIns(3,4)P 2 , Ins(3,4)P 2 , or Ins(1,3,4)P 3 (data not shown). Additional factors and/or processing may be required for the activation of these splice variants. Expression in mammalian cells may be required to characterize their enzymatic properties. We designate the isozymes with the hydrophilic C termini as types I␣ and II␣ 4-phosphatases and those with hydrophobic C termini as types I␤ and II␤ 4-phosphatases. Human EST clone yy41 g12 represents a partial clone of human 4-phosphatase II␤ that is 100% identical to the rat 4-phosphatase II␤ over the 51 amino acids predicted from sequence obtained from a BLAST search (13) of the GenBank TM data base indicating that the type II␤ splice variant is present in humans (data not shown).
FIG. 5. Northern blot analysis of the expression of human 4-phosphatase type II in various tissues. Lanes 1-8 are mRNA isolated from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas, respectively. The Northern blot was probed using the 362-base pair PCR product amplified from the 5Ј-end of human EST clone yy10a10. currently have been identified. The role of the 4-phosphatase isozymes in the regulation of PtdIns(3,4)P 2 -mediated signaling will be the focus of future studies.