lipase C to yield inositol phosphates and 1,2-diacylglycerol (6,. 7). In contrast, the ..... sodium phosphate, pH 7.0, 20 mM 2-mercaptoethanol, 4 pg each of.
THEJOURNALOF BIOLOGICAL CHEMISTRY (c)
Vol. 266, No. 27, Issue of September 25, pp. 18378-18386.1991 Printed in U.S.A.
1991 by The American Society for Biochemistry and Molecular Biology, Inc
Isolation and Characterizationof Two 3-Phosphatases That Hydrolyze Both Phosphatidylinositol 3-Phosphate and Inositol 1,3-Bisphosphate* (Received for publication, March 19, 1991)
Kevin K. CaldwellS, DanielL. Lips$, Vinay S. Bansal, and PhilipW. MajerusQ From the Divisionof Hematology-Oncology, Washington University School of Medicine, St. Louis, Missouri63110
Inositol-polyphosphate 3-phosphatase catalyzes the hydrolysis of the 3-position phosphate bond of inositol 1,3-bisphosphate (Ins(1,3)Pz) to form inositol l-monophosphate and inorganic phosphate (Bansal, V. S., Inhorn, R. C., and Majerus, P. W. (1987)J. Biol. Chem. 262,9444-9447). Phosphatidylinositol 3-phosphatase catalyzes the analogous reaction utilizing phosphatidylinositol 3-phosphate (PtdIns(3)P) as substrate to form phosphatidylinositol andinorganicphosphate (Lips, D. L., and Majerus, P. W. (1989)J. Biol. Chem. 264, 19911-19915). We now demonstrate that these enzyme activities are identical. Two forms of the enzyme, designated Type I and I1 3-phosphatases, were isolatedfrom rat brain. The Type I 3-phosphatase consisted of a protein doublet that migrated a t a relative M, of 65,000 upon sodium dodecyl sulfate (SDS)polyacrylamidegelelectrophoresis.The M, of this isoform upon size-exclusion chromatography was 110,000, suggesting that the native enzyme is a dimer. The Type I1 enyzme consisted of equal amounts of an M, = 65,000 doublet and an M, = 78,000 band upon SDS-polyacrylamide gel electrophoresis. This isoform displayed an M, upon size-exclusion chromatography of 147,000, indicating that it is a heterodimer. The Type I1 3-phosphatasecatalyzed the hydrolysis of Ins(1,3)Pzwith a catalytic efficiencyof one-nineteenth of that measuredfortheType I enzyme,whereas PtdIns(3)P was hydrolyzedby the Type I1 3-phosphatase at threetimes therate measured for the Type 13phosphatase. The M , = 65,000 subunits of the two forms of 3-phosphatase appear to be the same based on co-migration on SDS-polyacrylamide gelsand peptide maps generated with Staphylococcus aureus protease VS and trypsin. The peptide map of the M , = 78,000 subunit was different from that of the M , = 65,000 subunits. Thus, we propose that the differing relative specificities of the Type I and I1 3-phosphatases for Ins(1,3)Pz and PtdIns(3)Pare due to the presence of the M, = 78,000 subunit of the TypeI1 enzyme.
Lipid- and water-soluble molecules derived from phospha* This work was supported by Specialized Center for Research in Thrombosis Grant H L 14147, Grant HL 16634, and Training Grant H L 07088 from theNationalInstitutes of Health.Thecosts of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. $ These two authors contributed equally to this study. To whom correspondence should be addressed: Div. of Hematology-Oncology, Washingkon University School of Medicine, 660 S. Euclid Ave., Box 8125, St. Louis, MO 63110. Tel.: 314-362-8801; Fax: 314-362-8813.
tidylinositol (PtdIns)’ have been shown to actas signals coupling various extracellularstimuli to intracellularresponses (1-3). Five phosphorylated forms of theparent molecule, PtdIns, have been identified: phosphatidylinositol 4-phosphate (PtdIns(4)P), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), phosphatidylinositol 3-phosphate(PtdIns(3)P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3)(4, 5). Of these lipids, PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 are hydrolyzed by PtdIns-specific phospholipase C to yield inositol phosphates and 1,2-diacylglycerol(6, 7). In contrast, the 3-phosphate-containing lipids are resistant to theaction of PtdIns-specific phospholipase C (8,9). Rather, these lipids are metabolized by a group of relatively poorly described phosphatases and kinases (4, 5). We recently identified one of these enzymes, phosphatidylinositol 3-phosphatase, in NIH 3T3 cell extracts (10). This enzyme catalyzes the hydrolysis of the phosphate bond in the 3-position of the inositol ring of PtdIns(3)P to form PtdIns and inorganic phosphate. In addition to being derived directly from phosphatidylinositols, inositol phosphates are formed from other inositol phosphates by highly selective phosphatases and kinases (3). Over 20 inositol phosphates have been identified in various cells (3). Among these inositol phosphates is inositol 1,3bisphosphate (Ins(l,3)P2). Thismolecule is formed by inositol-polyphosphate 4-phosphatase-catalyzed hydrolysis of inositol 1,3,4-trisphosphate (11). Ins(l,3)P2 is degraded by inositol-polyphosphate3-phosphatase, which catalyzes the hydrolysis of the 3-positionphosphate to yield inositol 1monophosphate (Ins(1)P) and inorganic phosphate (11).We now demonstrate that inositol-polyphosphate 3-phosphatase and phosphatidylinositol3-phosphatase copurify from rat brain. Two forms of the active enzyme, designated Type Iand I1 3-phosphatases, were identified. EXPERIMENTALPROCEDURES
Materials-Phosphocellulose vine serum albumin (fraction pared without trichloroacetic sin, and phenylmethylsulfonyl
(medium mesh), octyl glucoside, bo-
V, globulin-free), cytochrome c (preacid), protein A-Sepharose CL-4B, trypfluoride were purchased from Sigma.
The abbreviations used are: PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4,5)Pz, phosphatidylinositol4,5-bisphosphate;PtdIn~(3,4,5)P:~,phosphatidylinositol 3,4,5-trisphosphate; Ins(l)P, inositol 1-monophosphate; Ins(l,3)P2, inositol 1,3-bisphosphate;Ins(1,3,4)P:j, inositol 1,3,4-trisphosphate; Ins(1,3,4,5)P4, inositol1,3,4,5-tetrakisphosphate; BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane;MES, 2 - ( N morpho1ino)ethanesulfonic acid; HEPES,N-2-hydroxyethylpiperazine-N”2-ethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitri1o)ltetraacetic acid; PMSF,phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PDGF, platelet-derivedgrowth factor; HPLC,high performance liquid chromatography.
18378
Type I and II3-Phosphatases Hydroxylapatite (Bio-Gel HTP), Dowex AG 1-X8 formate (200-400 mesh), SDS-PAGE molecular weight standards, gel filtration standards, and protein assaydye reagent were from Bio-Rad. AprotininandIns(1,3,4,5)P4 were purchasedfromBoehringer Mannheim. Leupeptin and calpain inhibitors I and I1 were supplied by both Boehringer Mannheim and Calbiochem. Benzamidine, pepstatin A, and Ins(1,3,4)P3 were purchasedfrom Calbiochem. ["HI In~(1,3,4)P:~, [:'H]Ins(1,3,4,5)P4, and[r-:"P]ATP (6000 Ci/mmol) were purchased from Du Pont-New England Nuclear. ['H]Ins(3,4)PZ was prepared by treating [:'H]Ins(1,3,4)P3 with purified inositolpolyphosphate 1-phosphatase as described (12). ''1 (protein iodination-grade) and Staphylococcus aureus protease V8 were from ICN (Costa Mesa, CA). All other chemicalswere from commercial sources. Preparation of ~"P]Ptdlns(3)P"NIH 3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium with 10% (v/v) calf serum. T h e medium was removed, and the cells were stimulated for 15 min at 37 "C withhumanplatelet-derived growth factor (PDGF) (BB homodimer form, 100 ng/ml) in Dulbecco's modified Eagle's medium containing 100 pg of bovine serum albumin/ml. The medium was removed; and the cells were washed with phosphate-buffered saline at 4 "C and then lysed with buffer that contained 50 mM NaC1, 20 mM Tris-HC1, pH 7.4, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM phenylmethylsulfonylfluoride, 200 p~ NaaV04, and 1%(v/v) Triton X-100 as described by Coughlin et al. (13) with slight modification. The lysate (500 pl/106 cells) was left a t 4 "C for 20 min, and particulate matter was removed by centrifugation a t 5000 X g for 10 min at 4 "C. Rabbit anti-mouse PDGF receptor antiserum (40 pl), kindly provided by V. Masakowski and T. Deuel (Washington University, St. Louis, MO), was added and mixed a t 4 "C for 2 h. Protein A-Sepharose CL-4B was added, and the mixture was rotated for an additional 1 h at 4"C. The protein A-Sepharose wascollectedby centrifugation andwashed three times with each of the following: 200 p~ Na:'VO, and 1% (v/v) Nonidet P-40 in phosphate-bufferedsaline; 500 mM LiC1, 100 mM Tris-HC1, pH 7.5, and 200 p~ Na:'VO,; and, finally, 100 mM NaCl, 10 mM Tris-HCI, pH 7.5, 1 mM EDTA, and 1 mM EGTA asdescribed by Serunian etal. (9) with slight modification. The washed immunoprecipitate was incubated for 60 min a t 37 "C with 200 p~ PtdIns, 5 p~ [r-:"P]ATP (800 Ci/mmol)in 20 mM HEPES, pH 7.5, 10 mM MgCl,, and 0.5 mM EGTA in a final volume of 125 pl. The ['"P]PtdIns(3)P product was extracted (14) and characterized (8, 10).Theproductcontained 90-95% ["'P]PtdIns(3)P, 2-10% [:"P]PtdIns(4)P, and0-2% ?'Pi. Assay of ["2P]Ptdlns(3)P3 Hydrolysis-PtdIns(3)P hydrolysis was measured as follows. ['2P]PtdIns(3)P in chloroform/methanol and PtdIns (carrier lipid) were dried under a stream of nitrogen; suspended in 500 mM KCl, 100 mM MES, p H 6.5, 12.5 mbf EDTA, and 1.5% (w/v) octyl glucoside; and sonicated for 30 s in a bath sonicator. Reaction mixtures contained 1 nM ["'PP]PtdIns(Y)P, 20 p M PtdIns, 100 mM KC1,20 mM MES,pH 6.5, 2.5 mM EDTA, 0.2 mg of cytochrome c/ml, and 0.3% (w/v) octyl glucoside in 20 pl. Reactions were started by addition of enzyme, incubated a t 37 "C for 3-10 min, and terminated by addition of 500 pl of 10% (w/v) trichloroacetic acid followed by 50 pl of 20% (v/v) Triton X-100 as described (15). The mixture was centrifuged, and the aqueous and organic phases were countedin a liquid scintillationcounterwithScintiVerse I (Fisher). Radioactivity intheproduct (""PO4) was 10-50%of the total. The limited quantitiesof [,"'P]PtdIns(3)P that could be synthesized precluded standard methods of enzyme characterization under conditions of zero-order kinetics (ie. [SI >> &). Instead, enzyme assays were performed under conditions of first-order kinetics (i.e. [SI
