J Biol Chem, Vol. 274, Issue 51, 36790-36795, December 17, 1999

Novel Inositol Polyphosphate 5-Phosphatase Localizes at Membrane Ruffles^*

Yasuhiro Mochizuki and Tadaomi Takenawa

From the Department of Biochemistry, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We have cloned a novel inositol polyphosphate 5-phosphatase from the rat brain cDNA library. It contains two highly conserved 5-phosphatase motifs, both of which are essential for its enzymatic activity. Interestingly, the proline content of this protein is high and concentrated in its N- and C-terminal regions. One putative SH3-binding motif and six 14-3-3 -binding motifs were found in the amino acid sequence. This enzyme hydrolyzed phosphate at the D-5 position of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and phosphatidylinositol 4,5-bisphosphate, consistent with the substrate specificity of type II 5-phosphatase, OCRL, synaptojanin and synaptojanin 2, already characterized 5-phosphatases. When the Myc-epitope-tagged enzyme was expressed in COS-7 cells and stained with anti-Myc polyclonal antibody, a signal was observed at ruffling membranes and in the cytoplasm. We prepared several deletion mutants and demonstrated that the 123 N-terminal amino acids (311-433) and a C-terminal proline-rich region containing 277 amino acids (725-1001) were essential for its localization to ruffling membranes. This enzyme might regulate the level of inositol and phosphatidylinositol polyphosphates at membrane ruffles.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Inositol and phosphatidylinositol polyphosphates play important roles in a variety of signal transduction systems. Therefore, intracellular levels of these second messenger molecules are thought to be tightly controlled and promptly changed by the enzymes in response to extracellular stimuli. Inositol polyphosphate 5-phosphatase is the enzyme that specifically hydrolyzes phosphate at the D-5 position of inositol or phosphatidylinositol polyphosphates and has been conserved from yeast to human. Seven different enzymes and numerous splicing isoforms have been isolated in mammals (1-15). All 5-phosphatases possess two highly conserved catalytic motifs and are classified into three groups based on their substrate specificity. 1) Type I 5-phosphatase; this enzyme hydrolyzes only water-soluble substrates such as Ins(1,4,5)P₃¹ and Ins(1,3,4,5)P₄ (3, 16). 2) SHIP 1 (for SH2 containing inositol polyphosphate 5-phosphatase 1) and SHIP 2; SHIP 1 selectively dephosphorylates Ins(1,3,4,5)P₄ and PtdIns(3,4,5)P₃ that contain phosphate at the D-3 position of the inositol ring (5, 7, 8). SHIP 2 hydrolyzes PtdIns(3,4,5)P₃, but its Ins(1,3,4,5)P₄ phosphatase activity has not been confirmed (17, 18). 3) Type II 5-phosphatase, OCRL, synaptojanin and synaptojanin 2; these enzymes exhibit broad substrate specificity. They hydrolyze water-soluble substrates such as Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄ and lipid substrates such as PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ (6, 11, 12, 16, 19-22). Synaptojanin 2 is a recently discovered 5-phosphatase, and its catalytic motifs are identical to synaptojanin (11, 12). OCRL was identified as a causative gene of Low's oculocerebrorenal syndrome, an X chromosome-linked developmental disorder (2). OCRL showed a strong preference for lipid substrate (16). The cells derived from the Low's oculocerebrorenal syndrome patient were defective in OCRL activity and accumulated 2-3-fold more PtdIns(4,5)P₂ than normal cells (20).

There is evidence that phosphatidylinositol polyphosphates such as PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ play important roles in the regulation of the actin cytoskeleton (22-24). PtdIns(4,5)P₂ binds to actin-binding proteins such as vinculin, -actinin, profilin, and gelsolin, and promotes actin filament formation (25-27). Of the seven distinct 5-phosphatases, only synaptojanin has been demonstrated to hydrolyze PtdIns(4,5)P₂ bound to actin regulatory proteins such as vinculin,  -actinin, and profilin in vitro (22). In addition, it has been shown that PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ modulate the function of various proteins such as protein kinase C (28), phospholipase D (29, 30), protein kinase B/Akt (31), ATP-sensitive potassium channel (32-34), and ADP-ribosylation factor (35). Therefore, it is plausible that lipid phosphatase, like OCRL and synaptojanin, hydrolyzes those phosphatidylinositol polyphosphates and plays an important role in the actin depolymerization mechanism as well as other functions.

Here, we report the cloning and characterization of a novel 5-phosphatase. This enzyme dephosphorylates the D-5 position of inositol and phosphatidylinositol polyphosphates at ruffling membranes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Molecular Cloning of the Rat Novel Inositol Polyphosphate 5-Phosphatase-- A partial human cDNA clone (GenBank^TM accession number H14886) was obtained from Genome System, Inc. (St. Louis, MO). H14886 is a 468-base pair human cDNA fragment, and its deduced amino acid sequence contains a 5-phosphatase catalytic motif2-like sequence. The fragments were labeled by random hexamer priming and used to screen a ZAPII rat brain cDNA library (Stratagene, La Jolla, CA). Positive clones were subcloned into pBluescript SK() by an in vivo excision method and sequenced. Complete sequence data was obtained from both strands using a conventional dideoxy-termination method.

Northern Blot Analysis-- Membranes containing mRNA (2 µg of poly(A) RNA was contained in each lane) were purchased from OriGene Technology, Inc. (Rockville, MD). Total RNA was isolated from cultured cells using MagExtractor-RNA- and MagExtractor System (TOYOBO Co., Ltd., Osaka, Japan), and approximately 10 µg of the RNA was blotted on a nylon membrane. The sequence of rat novel 5-phosphatase specific region, nucleotides 2362-3000, was amplified by polymerase chain reaction and used as an [-³²P]dCTP-labeled 0.7-kb cDNA probe. Hybridization was performed following the protocol of OriGene Technology, Inc., and the probe was hybridized at 42 °C.

Cell Culture and Expression of Recombinant Proteins in COS-7 Cells-- COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Nissui, Tokyo, Japan) containing 10% fetal calf serum and 60 µg/ml kanamycin, and kept at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Rat novel 5-phosphatase cDNA, its deletion mutants and partial human SHIP 1 cDNA (nucleotides 1461-4079 of human SHIP 1 cDNA; GenBank^TM accession number U57650) were subcloned into the eukaryotic pCMV6-Myc expression vector. Constitutively active Rac1, Rac1G12V construct, was ligated into the eukaryotic pEF-BOS-FLAG expression vector. All constructs were transfected into COS-7 cells by a conventional electroporation method, and cells were harvested or fixed after 48 h (22). The expression and the size of expressed proteins were checked by immunoblotting.

Dephosphorylation of Novel Inositol Polyphosphate 5-Phosphatase Protein-- Recombinant novel 5-phosphatase expressing cells (1 × 10⁶ cells/60-mm tissue culture dish (Falcon)) were washed once with PBS() and harvested with 200 µl of the reaction buffer for alkaline phosphatase (50 mM Tris-HCl, pH 7.5, 1 mM MgCl₂, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). After a brief sonication, the cell lysate was incubated for 10 min at 30 °C. Then 100 µl of the lysate with or without 30 units of calf intestine alkaline phosphatase (Takara Shuzo Co., Ltd., Biomedical Group, Shiga, Japan) was further incubated for 15 min at the same temperature. The mixture was analyzed by immunoblotting.

Enzyme Assay-- Cells (7 × 10⁶ cells/150-mm tissue culture dish (Falcon)) transfected with Myc-epitope-tagged novel 5-phosphatase construct or empty vector were cultured for 2 days and harvested with 1 ml of cold lysis buffer (40 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). The cells were briefly sonicated and centrifuged at 10,000 × g for 20 min at 4 °C. Supernatant was collected and rotated with 25 µl of anti-Myc monoclonal antibody (Santa Cruz Biotechnology, Inc.) for 1 h at 4 °C. Then protein A beads (Pierce) (50 µl) were added, and the solution was rotated for 1 h. After that, the beads were washed with lysis buffer five times and finally suspended in buffer for inositol polyphosphates or PtdIns(4,5)P₂ phosphatase assay.

Assay of inositol polyphosphate 5-phosphatase activity was carried out as described by Connolly et al. (36) using [³H]Ins(1,3,4)P₃, [³H]Ins(1,4,5)P₃, and [³H]Ins(1,3,4,5)P₄. The separation of inositol polyphosphates by high performance liquid chromatography was done according to the method of Zhang and Buxton (37). The flow rate was 0.6 ml/min, and each fraction was collected for 30 s. Collected samples were diluted to 1/20 volume with distilled water and quantitated by liquid scintillation counting. For the assay of Ins(1,3,4)P₃ hydrolyzing activity, the D-5 position phosphate of Ins(1,3,4,5)P₄ was hydrolyzed by recombinant SHIP 1 protein and used as a substrate. The SHIP 1 protein was prepared the same way as the novel 5-phosphatase.

The PtdIns(4,5)P₂ phosphatase activity was determined (38). The spots on the thin layer chromatography plate were visualized by exposing the plate to x-ray film (Eastman Kodak Scientific Co., Rochester, NY) for 6 days at 80 °C. Before the exposure, the intensity of the radioactivity was enhanced by EN³HANCE spray (NEN Life Science Products).

Cell Staining-- Transfected cells cultured on glass coverslips were fixed with 10% formaldehyde in PBS() for 10 min at room temperature and washed three times with PBS(). Then they were incubated for 5 min in 0.2% Triton X-100 in PBS() at room temperature and washed with PBS() five times. The cells were further incubated with anti-Myc polyclonal antibody (Santa Cruz Biotechnology, Inc.) for 1 h. After five washes with PBS(), the cells were incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (ICN Pharmaceuticals, Inc., Costa Mesa, CA) and rhodamine-phalloidin (Molecular Probes, Eugene, Oregon) for 30 min. When active Rac was co-transfected, anti-FLAG monoclonal antibody (Eastman Kodak Scientific Co.) and anti-Myc polyclonal antibody were used as primary antibodies and rhodamine-conjugated anti-mouse IgG antibody (ICN Pharmaceuticals, Inc.) was substituted with rhodamine-phalloidin. The cells were mounted in glycerin-PBS() and observed with a fluorescence microscope.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

cDNA Cloning of the Novel Inositol Polyphosphate 5-Phosphatase-- The predicted amino acid sequence of human EST clone H14886 is very similar to OCRL and synaptojanin, and contains the 5-phosphatase catalytic motif2-like sequence (2, 6). Therefore, we regarded H14886 as a partial cDNA fragment of a novel 5-phosphatase. Using H14886 as a probe, we obtained several cDNA clones from a rat brain cDNA library and isolated a 3,322-base pair rat putative 5-phosphatase cDNA (Fig. 1). Our rat cDNA clone contained the polyadenylation signal AATTAAA (nucleotides 3304-3310) (Fig. 1). The same polyadenylation signal and poly(A) sequence are found in another 1496-base pair human EST clone, U45975. There was no in-frame upstream stop codon in our cDNA clone. However, we concluded that the ATG underlined in Fig. 1 is the initiation codon for rat putative 5-phosphatase mRNA because 1) the sequence around the first putative ATG codon agrees with Kozak's consensus rule (GCAGACATGG versus GCC(G/A)CCATGG) (39), and 2) Northern blot analysis indicated that the rat putative 5-phosphatase mRNA is 3.4-kb long, which is nearly identical to the length of our cDNA clone (Fig. 4). Searches of the current GenBank^TM data base with BLASTN algorithm revealed that part of synaptojanin and synaptojanin 2 are homologous to the 1900-2040-base pair region of the rat putative 5-phosphatase cDNA sequence (60 and 69%, respectively), which includes the 5-phosphatase catalytic motif2.

View larger version (79K): [in this window] [in a new window]   Fig. 1.   Nucleotide and corresponding amino acid sequence of PIPP cDNA. The proline residues are indicated by shaded boxes. Characteristic amino acid sequences of 5-phosphatase are boxed. The putative ATG start codon, TGA stop codon and AATTAAA polyadenylation signal are underlined. The amino acid numbers are indicated in the right margin, and nucleotide numbers in the left margin. The N-terminal proline-rich region is from amino acid 1 to 433 and the C-terminal region is from amino acid 866 to 1001. A putative SH3-binding motif is located in the N-terminal proline-rich region (*). Six motifs similar to the RSXSXP 14-3-3 -binding motif are indicated by lines with two arrowheads.

Features of the Amino Acid Sequence of the Novel Inositol Polyphosphate 5-Phosphatase and Its Recombinant Protein-- The putative amino acid sequence of the 5-phosphatase cDNA indicates that it is a proline-rich 5-phosphatase with a molecular mass of 107 kDa (Fig. 1). Usually, the average proline content of proteins in eukaryotes is 4-5% (40), but that of putative 5-phosphatase is 13.3% and concentrated in its N- and C-terminal regions (Fig. 1). The N-terminal proline-rich region contains 21.0% and the C-terminal region 18.4% proline. In contrast, type I 5-phosphatase contains 3.9% proline, SHIP 1, 8.0%; SHIP 2, 9.3%; type II 5-phosphatase, 4.5%; OCRL, 6.2%; synaptojanin, 7.0%; and synaptojanin 2, 4.5%. Therefore, we designated the putative 5-phosphatase as PIPP (proline-rich inositol polyphosphate 5-phosphatase). The N-terminal proline-rich region contained one putative SH3-binding motif, PRSPSR (Fig. 1) (41, 42). Recently, it was reported that type I 5-phosphatase was activated when it bound to platelet protein, pleckstrin, or 14-3-3  (43, 44). Campbell et al. (44) showed that type I 5-phosphatase bound to the 14-3-3  via its RSXSXP motif (RSESEE). We found six such motifs in the N- and C-terminal proline-rich regions of PIPP (Fig. 1). PIPP contains the largest number of 14-3-3 -binding motifs of the known 5-phosphatases. Therefore, the enzymatic activity of PIPP may be elevated by binding to 14-3-3 . Fig. 2 shows the catalytic motifs of PIPP that are conserved in other 5-phosphatases. Within the two motifs, the amino acids shown to be essential for 5-phosphatase activity are indicated by asterisks (45, 46) and are present in PIPP.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Comparison of characteristic amino acid sequences of inositol polyphosphate 5-phosphatases with that of PIPP. Two highly conserved motifs of 5-phosphatase are aligned with PIPP. Asterisks (*) indicate the amino acids shown to be important for 5-phosphatase activity (45, 46). The enzymes are classified by specificity for substrates. Type I 5-phosphatase hydrolyzes Ins(1,4,5)P3 and Ins(1,3,4,5)P4. SHIP 1 and SHIP 2 react with the substrates that contain phosphate at the D-33 position of the inositol ring, such as PtdIns(3,4,5)P3. Type II 5-phosphatase, OCRL, and synaptojanin hydrolyze Ins(1,4,5)P3, Ins(1,3,4,5)P4, PtdIns(4,5)P2, and PtdIns(3,4,5)P3. The GenBankTM accession numbers are as follows: type I (X75094); SHIP 1 (U57650); SHIP 2 (Y14385); type II (M74161); OCRL (M88162); synaptojanin (U45479).

When recombinant PIPP protein was expressed in COS-7 cells and analyzed by immunoblotting, two bands were detected (Fig. 3). Treatment of cell lysate with alkaline phosphatase reduced the upper band indicating that a part of the ectopically expressed PIPP is phosphorylated. The molecular mass of PIPP was estimated at about 107 kDa from its amino acid sequence but the dephosphorylated PIPP still seemed slightly larger in SDS-polyacrylamide gel electrophoresis (Fig. 3). This may be due to the proline-rich sequence of PIPP (40).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Immunoblotting of PIPP recombinant protein. Recombinant PIPP was expressed in COS-7 cells and detected by immunoblotting with anti-Myc polyclonal antibody (left). Cell lysate incubated without calf intestine alkaline phosphatase (CIAP) for 15 min at 30 °C (middle), and with calf intestine alkaline phosphatase (right).

Our results suggest that recombinant PIPP is highly phosphorylated in the cytoplasm. But Western blot analysis revealed that anti-phosphotyrosine antibody (PY20) did not stain phosphorylated PIPP (data not shown). Therefore, PIPP is probably phosphorylated at serine/threonine residues, which might regulate the activity.

Northern Blot Analysis-- The results of Northern blot analysis are shown in Fig. 4. PIPP was expressed in brain, heart, kidney, stomach, small intestine, and lung. The size of the mRNA was estimated at 3.4 kb in those tissues. In spleen, thymus, skeletal muscle, testis, and skin, no signal was observed. All cultured cells examined expressed PIPP, but Jurkat and HL-60 cells showed an especially high PIPP mRNA content. Only liver showed the 2.4-kb band. This signal might indicate the existence of a splicing isoform of the PIPP or another undiscovered 5-phosphatase in liver.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Northern blot analysis of PIPP. Rat PIPP-specific 0.7-kb cDNA probe detected an approximately 3.4-kb band. The numbers depicted on the left are molecular weight markers (left and middle panels) and positions of ribosomal RNA (right panel). C6, rat glioma cells; A431, human epidermoid carcinoma cells; C2C12, mouse myoblast cells; NIH 3T3, mouse fibroblast cells; NIH 3T3(Ras), Ras-transformed NIH 3T3 cells; COS-7 cells, simian kidney cells; Jurkat, human leukemic T cells; HL-60, human myeloblastic leukemia cells.

Enzymatic Activity-- Myc-epitope-tagged PIPP was expressed in COS-7 cells and purified by immunoprecipitation using anti-Myc monoclonal antibody. When Ins(1,4,5)P₃ was incubated with recombinant PIPP and the mixture was analyzed by high performance liquid chromatography, two peaks were observed (Fig. 5A). The retention time of these two peaks indicated that the former was inositol bisphosphate and the latter was substrate, Ins(1,4,5)P₃ (37). PIPP hydrolyzed Ins(1,4,5)P₃ to inositol bisphosphate and also removed one phosphate from Ins(1,3,4,5)P₄ and produced inositol trisphosphate (Fig. 5B). To determine which phosphate was hydrolyzed by PIPP, Ins(1,3,4)P₃ was incubated with PIPP, but no hydrolysis was observed in this case (Fig. 5C). These results indicate that PIPP specifically hydrolyzes phosphate at the D-5 position in Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Inositol polyphosphate 5-phosphatase activity of PIPP. Hydrolysis of Ins(1,4,5)P3 (A) and Ins(1,3,4,5)P4 (B) by recombinant PIPP. PIPP, Myc-epitope-tagged PIPP protein was purified by immunoprecipitation and incubated with substrate (). Mock, cells containing the Myc-epitope vector pCMV6-Myc were treated in the same manner (). Recombinant PIPP or mock was incubated with each substrate for 15 min, and the reaction mixture was analyzed by high performance liquid chromatography. When Ins(1,3,4)P3 was used as a substrate, no hydrolysis was observed (C).

The catalytic motifs of PIPP are very similar to OCRL and synaptojanin. These 5-phosphatases hydrolyze inositol polyphosphates as well as PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ (16, 19-22). We also checked the lipid phosphatase activity of PIPP. As shown in Fig. 6, when [³H]PtdIns(4,5)P₂ was incubated with PIPP, [³H]phosphatidylinositol monophosphate was formed. When the phosphate at the D-5 position of PtdIns(4,5)P₂ was labeled with ³²P and incubated with PIPP, [³²P]phosphatidylinositol monophosphate was not formed (data not shown), showing that PIPP has lipid phosphatase activity and can hydrolyze phosphate at the D-5 position of PtdIns(4,5)P₂. Thus, according to the substrate specificity, PIPP is classified with type II 5-phosphatase, OCRL, synaptojanin, and synaptojanin 2 (21).

View larger version (59K): [in this window] [in a new window]   Fig. 6.   Lipid phosphatase activity of PIPP. [3H]PtdIns(4,5)P2 was incubated with PIPP or mock, and the reaction product was analyzed by thin layer chromatography. In the PIPP lane, PtdIns(4,5)P2 hydrolysis was observed, but no hydrolysis was observed when mock was incubated with PtdIns(4,5)P2.

Overexpression of PIPP and Its Deletion Mutants in COS-7 Cells-- Myc-epitope-tagged PIPP was expressed in COS-7 cells, and its cellular localization was revealed by anti-Myc polyclonal antibody. As indicated in Fig. 7A, some PIPP was clearly condensed at the cell periphery and some dispersed in the cytoplasm. The same cells were stained with rhodamine-phalloidin to visualize the actin cytoskeleton (Fig. 7B). Some PIPP was localized in cortical areas with the actin filaments locating at ruffling membranes. However, alterations in the actin cytoskeleton were not observed, in contrast to reports on overexpression of OCRL and synaptojanin in cells (22, 47).

View larger version (66K): [in this window] [in a new window]   Fig. 7.   Expression of recombinant PIPP in COS-7 cells. Recombinant PIPP was expressed in COS-7 cells and visualized with anti-Myc polyclonal antibody and fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (A). The same cells were stained with rhodamine-phalloidin (B).

We prepared several deletion mutants of PIPP to determine the region essential for its localization to ruffling membranes and co-expressed it with constitutively active Rac1 mutant in COS-7 cells (Fig. 8, panel I). Constitutively active Rac1 mutant co-localizes with actin filaments at ruffling membranes and accelerates membrane ruffling (48, 49). Apparently, PIPP co-localized with the active Rac1 mutant (Fig. 8, panel II, A and B) confirming that PIPP localizes at ruffling membranes. Mut2 and Mut3 exhibited the same localization pattern as the full-length of PIPP (data not shown). Truncated PIPP without the N-terminal proline-rich region (Mut4) located to membrane ruffles at a much lower intensity than wild type or mutants with partial proline-rich regions (Mut2 and Mut3, Fig. 8, panel II, C and D). When the C-terminal proline-rich region containing 277 amino acids (725-1001) was deleted (Mut6), the localization of the enzyme to the ruffling membranes was abolished (Fig. 8, panel II, E and F). Mut5 did not contain either the N- or C-terminal proline-rich regions and did not localize to ruffling membranes (Fig. 8, panel II, G and H). These results indicate that the C-terminal proline-rich region from residues 725 to 1001 is essential for the localization of PIPP to ruffling membranes and a part of the N-terminal proline-rich region from residues 311 to 433 contributes to the localization. PIPP contains one putative SH3-binding motif and five 14-3-3 -binding motifs in the proline-rich regions that are involved in its cellular distribution. The identification of PIPP-binding proteins should deepen the understanding of the function of PIPP.

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Cellular localization of PIPP and its deletion mutants in COS-7 cells. Panel I, structure of recombinant PIPP and its deletion mutants. All constructs had an N-terminal Myc-epitope tag (gray box). N- and C-terminal proline-rich regions are indicated by shaded boxes. The two 5-phosphatase catalytic motifs are indicated by black boxes. The amino acid number of the deleted points are indicated at the top. Panel II, cellular localization of PIPP and its deletion mutants. Wild type (A and B); Mut4 (C and D); Mut6 (E and F); Mut5 (G and H), and Mut1 (I and J). Recombinant PIPP and its deletion mutants were stained with anti-Myc polyclonal antibody and fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (A, C, E, G, and I). Active Rac1 mutant was detected with anti-FLAG monoclonal antibody and rhodamine-conjugated anti-mouse IgG antibody (B, D, F, H, and J).

Mut1 did not contain the two catalytic motifs of 5-phosphatase localized to ruffling membranes (Fig. 8, panel II, I and J). Membrane ruffling, induced by constitutively active Rac1 mutant, was not affected by the co-expression of any deletion mutants of PIPP (Fig. 8, panel II, B, D, F, H, and J). These results indicate that PIPP does not participate in the re-organization of the actin cytoskeleton but may be involved in modulation of the function of inositol and phosphatidylinositol polyphosphate-binding proteins that are present at membrane ruffles.

In summary, we have cloned a novel 5-phosphatase from a rat brain cDNA library. It is a proline-rich protein and hydrolyzed the D-5 position of phosphate in Ins(1,4,5)P₃, Ins(1,3,4,5)P₄ and PtdIns(4,5)P₂. Therefore, we designated this novel 5-phosphatase as PIPP. PIPP is localized at membrane ruffles and may be involved in the modulation of the function of proteins that are present at membrane ruffles.

	FOOTNOTES

^* This work was supported in part by a grant-in-aid for Research for the Future Program from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 81-3-5449-5508; Fax: 81-3-5449-5417; E-mail: takenawa@ims.u-tokyo.ac.jp.

	ABBREVIATIONS

The abbreviations used are: Ins(1, 4,5)P₃, inositol 1,4,5-trisphosphate; kb, kilobase; PBS(), phosphate-buffered saline without Ca²⁺ and Mg²⁺; TLC, thin layer chromatography; Ins(1, 3,4)P₃, inositol 1,3,4-trisphosphate; Ins(1, 3,4,5)P₄, inositol 1,3,4,5-tetrakisphosphate; PtdIns(4, 5)P₂, phosphatidylinositol 4,5-bisphosphate; PtdIns(3, 4,5)P₃, phosphatidylinositol 3,4,5-trisphosphate; EST, expressed sequence tag data base; SHIP 1, SH2 containing inositol polyphosphate 5-phosphatase 1.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES