Identification and Characterization of a Novel Inositol Polyphosphate 5-Phosphatase*

We have identified a cDNA encoding a novel inositol polyphosphate 5-phosphatase. It contains two highly conserved catalytic motifs for 5-phosphatase, has a molecular mass of 51 kDa, and is ubiquitously expressed and especially abundant in skeletal muscle, heart, and kidney. We designated this 5-phosphatase as SKIP (Skel- etal muscle and Kidney enriched Inositol Phosphatase). SKIP is a simple 5-phosphatase with no other motifs. Baculovirus-expressed recombinant SKIP protein ex-hibited 5-phosphatase activities toward inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol (PtdIns) 4,5-bisphosphate, and PtdIns 3,4,5-trisphosphate but has 6-fold more substrate specificity for PtdIns 4,5-bisphosphate ( K m 5 180 m M ) than for inositol 1,4,5-trisphosphate ( K m 5 1.15 m M ). The ectopic expression of SKIP protein in COS-7 cells and immuno-staining of neuroblastoma N1E-115 cells revealed that SKIP is expressed in cytosol and that loss

We have cloned a cDNA encoding a protein homologous to known IP5Pases and containing highly conserved amino acids indicative of a 5-phosphatase. This enzyme acts to hydrolyze D-5 positions of inositol polyphosphate and phospholipids. Expression of this enzyme causes the loss of actin stress fibers, suggesting a role in negatively regulating the actin cytoskeleton.

EXPERIMENTAL PROCEDURES
Materials-Inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate were purchased from Sigma. [  H14886 is a 468-bp human cDNA clone whose putative amino acid sequence contains an IP5Pase-conserved catalytic motif 2-like sequence. The human testis ZAPII cDNA library (Stratagene) was screened using this cDNA as a probe. All DNA probes were 32 P-labeled using a random primer labeling kit (Takara Shuzo) following the manufacturer's protocol.
Northern Blot Analysis-A membrane containing mRNA (2 g of poly(A) was contained in each lane) (human 12-lane multiple tissue Northern (MTN) blot) was pruchased from CLONTECH. The 1.9-kilobase pair human IP5Pase cDNAs including the coding region of SKIP were 32 P-labeled using a random primer labeling kit (Takara Shuzo) and probed with a following the manufacturer's protocol.
Bacterial Expression of Recombinant SKIP-The bacterial expression vector for human SKIP was constructed by ligating the 990-bp HindIII fragment, corresponding to amino acid residues 137-448, into His 6 -tagged pQE-30 bacterial expression vector (Qiagen). His-SKIP recombinant protein was expressed and purified on Ni 2ϩ -nitrilotriacetic acid-agarose beads as described by the manufacturer.
Antibodies-Polyclonal anti-SKIP antibodies were made by immunizing rabbits with the recombinant His 6 -tagged human SKIP. The * 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.
antibodies were affinity-purified from sera by conjugating the antibody proteins to CNBr beads (Amersham Pharmacia Biotech). The anti-Myc polyclonal antibody was purchased from Santa Cruz Biotechnology.
Baculovirus Expression and Purification of Recombinant SKIP-The cDNA constructs, GST-tagged full-length SKIP (GST-SKIP), and GSTtagged partial human SHIP 1 cDNA (nucleotides 1,461-4,079; Genbank TM accession number U57650) (GST-SHIP) were constructed by subcloning BglII-BamHI site polymerase chain reaction products encoding full-length GST amplified from pGEX-2T into the BamHI site of pFASTBAC1 (Life Technologies, Inc.) and then introducing either an EcoRI site full-length human SKIP fragment amplified by polymerase chain reaction or a BamHI site partial human SHIP 1. GST, GST-SKIP, GST-SHIP, and phosphatidylinositol 3-kinase catalytic subunit p110␣ proteins were expressed in Sf9 cells by infecting them with 1 ml of 1 ϫ The genome DNA sequence of human SKIP was obtained from the human genome data base (Homo sapiens chromosome 17, clone hRPK.107_N_19, Gen-Bank TM /EBI Data Bank accession number AC006405). Clone 2 has a 230-bp insertion corresponding to exon II, and Clone 3 has a 937-bp deletion in the Cterminal non-coding region (exon XIII). Positions of the start methionine and stop codon of each clone are indicated by asterisks. B, nucleotide and predicted amino acid sequences of human SKIP cDNA Clone 1. The nucleotide and amino acid numberings for human SKIP are indicated on the right. The amino acid numbering indicates the position relative to the putative start methionine. The inframe stop codon and the position of insertion in Clone 2 are indicated (underlined and vertical line, respectively), and conserved motifs 1 and 2 of IP5Pase are highlighted as white on black. A 937-bp deleted sequence in Clone 3, corresponding to exon XIII, is shaded. C, nucleotide and putative amino acid sequences of human SKIP cDNA Clone 2. The nucleotide and amino acid numberings are indicated on the right. The in-frame stop codon is underlined, and a 230-bp inserted sequence is shaded. 10 9 units/ml recombinant virus and cultured in Sf-900 serum-free medium at 28°C for 48 h.
Cell Culture and Expression of Recombinant Proteins in COS-7 Cells-COS-7 cells and N1E-115 cells were cultured in Dulbecco's modified Eagle's medium (Nissui Seiyaku) containing 10% fetal calf serum and 50 g/ml kanamycin in an atmosphere of 5% CO 2 at 37°C. Fulllength human SKIP was subcloned into the SR␣-and the Myc-tagged pCMV5 mammalian expression vector. These constructs (10 g) were transfected into COS-7 cells (1 ϫ 10 7 ) by the electroporation method. Cells were cultured for 24 h after transfection in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and then cultured 24 h in a serum-deprived medium before cells were harvested or fixed. 50 g/ml human recombinant epidermal growth factor (Life Technologies, Inc.) or 1% fetal calf serum was added after serum starvation for 24 h to analyze the effect. N1E-115 cells were differentiated into neuriteextended cells by culturing in the serum-free medium for 48 h and fixed.
Immunofluorescence of SKIP-Cells cultured on coverslips were fixed with 3.7% formaldehyde in Ca 2ϩ -and Mg 2ϩ -free phosphate-buffered saline and permeabilized with 0.2% Triton X-100 in Ca 2ϩ -and Mg 2ϩfree phosphate-buffered saline for 5 min. The cells were incubated with anti-SKIP polyclonal antibody or anti-Myc polyclonal antibody and visualized by a second antibody or rhodamine-conjugated phalloidin. The cells were observed with a confocal fluorescence microscope.

RESULTS AND DISCUSSION
cDNA Cloning of a Novel Inositol Polyphosphate 5-Phosphatase-In an attempt to identify novel inositol polyphosphate 5-phosphatases, we screened several cDNA libraries using human expressed sequence tag clone H14886 containing the 5-phosphatase catalytic motif 2-like sequence (see Fig. 2) as a probe (10,14). We obtained two novel cDNA clones encoding 5-phosphatase-like sequences. One is PIPP, a recently isolated 107-kDa protein (24). The other encodes a putative 5-phosphatase cDNA clone obtained from a human testis cDNA library and 87% identical to the mouse cDNA clone U96724. Three alternative splicing isoforms were obtained (Fig. 1A). Clone 1 is a 2,786-bp cDNA with an open reading frame predicting a protein with a molecular mass of 51 kDa (Fig. 1B). Clone 2 has a 230-bp insertion, corresponding to exon II, in the N terminus of Clone 1 with a putative molecular mass of 43 kDa (Fig. 1, A  and C). Clone 3 has a 937-bp deletion, corresponding to exon XIII, in the C-terminal non-coding region (Fig. 1B, shaded). In each splicing isoform, the open reading frame begins at the consensus start methionine with an upstream in-frame stop codon (Fig. 1, B and C, respectively) (31). This IP5Pase has two conserved catalytic motifs of IP5Pases (Fig. 2). To other IP5Pases, PIPP has a lot of similarity (24), whereas Type I phosphatase and Type III phosphatase SHIP have little resemblance. The full length of this IP5Pase also has greater identity with PIPP (53.3%) than do other IP5Pases (about 45%, see Fig. 2). However, no other motifs for interactions with other molecules are observed.
Northern Blot Analysis-The results of Northern blot anal-ysis of the expression of the human novel IP5Pase are shown in Fig. 3. Two different transcripts of 2.0 kilobases and about 3.0 kilobases, respectively, are detected. The longer ones are the transcripts of Clone 1 and Clone 2, and the 2.0-kilobase transcript is that of Clone 3. This IP5Pase is expressed in all the tissues we tested, but its expression is especially high in heart, skeletal muscle, and kidney. Thus we designated this IP5Pase SKIP. Only the 3.0-kilobase transcript was detected in the brain. Splicing Isoform Is Specifically Expressed According to the Type of Cell-To characterize the endogenously expressed SKIP protein, we generated a specific antibody against SKIP and blotted lysates from different tissues and cells (Fig. 4A). In addition to the 51-kDa form, a smaller 43-kDa band was detected. In rat brain lysate and mouse neuroblastoma N1E-115 cells, only the 51-kDa form was expressed. In contrast, in mouse C2C12 myoblast cells, C3H/10T1/2 cells, and Swiss 3T3 fibroblast cells, only the 43-kDa band was detected. Surprisingly, although SKIP cDNA was obtained from the human testis library, its expression was very weak in rat testis lysate.
To confirm that the 43-kDa band is really an alternatively spliced isoform, we subcloned the full length of Clone 1 and 2 into SR␣ expression vector, and then they were expressed in COS-7 cells. As shown in Fig. 4B, in addition to the 51-kDa form, endogenously expressed in COS-7 cells, the 43-kDa band was detected only in cells expressing Clone 2. This indicates that the 43-kDa band is indeed an alternative splicing isoform of SKIP.
To analyze the substrate specificity of SKIP, we determined the apparent K m values of the 5-phosphatase by Lineweaver-Burk plot analysis, using purified recombinant protein (Fig. 7). K m values of the PtdIns(4,5)P 2 and Ins(1,4,5)P 3 5-phosphatase are 180 M and 1.15 mM, respectively. This K m value for PtdIns(4,5)P 2 is comparable with that of other Type II 5-phosphatases, such as the 75-kDa Ins(1,4,5)P 3 5-phosphatase (K m ϭ 250 M) (28). SKIP hydrolyzes PtdIns(4,5)P 2 with a 6-fold higher affinity than for Ins(1,4,5)P 3 , indicating that SKIP functions as a 5-phosphatase that has high specificity for PtdIns(4,5)P 2 and probably also for PtdIns (3,4,5)  anti-Myc polyclonal antibody. As shown in Fig. 8, SKIP was localized at the cytoplasm and concentrated at the periphery of the nucleus. Actin filaments are also observed using rhodamine-phalloidin. COS-7 cells develop actin stress fibers throughout, but in SKIP-expressing cells, stress fibers disappeared from places where SKIP concentrated. This result suggests that SKIP has a negative effect on the formation of actin stress fibers, as synaptojanin 1 does (8). However, when COS-7 cells expressing the 51-kDa form of SKIP were stimulated with epidermal growth factor and fetal calf serum, no change in the localization of SKIP or actin stress fiber formation was observed (data not shown), indicating that SKIP does not function downstream of the mitogenic signals, whereas synaptojanin 1 associates with the epidermal growth factor receptor through Ash/Grb2 and acts downstream of it (8).
Then, to determine the endogenous expression of SKIP, rat neuroblastoma N1E-115 cells (expressing only the 51-kDa form) were immunostained with a specific antibody against SKIP (Fig. 9). In undifferentiated N1E-115 cells, staining was seen throughout except in the actin bundles at the cell periphery. In neurite extended cells, SKIP was expressed also in the neurites. Some strong spots were seen in the neurites, where actin staining was reduced (Fig. 9, C and D, arrowheads).