Phosphatidylinositol-4-phosphate 5-Kinase Isozymes Catalyze the Synthesis of 3-Phosphate-containing Phosphatidylinositol Signaling Molecules*

(Received for publication, March 18, 1997)

Xiaoling Zhang Dagger §, Joost C. Loijens §par , Igor V. Boronenkov **, Gregory J. Parker par , F. Anderson Norris Dagger , Jian Chen Dagger Dagger , Oliver Thum Dagger Dagger , Glenn D. Prestwich Dagger Dagger , Philip W. Majerus Dagger and Richard A. Anderson par **§§

From the Dagger  Division of Hematology-Oncology, Departments of Internal Medicine and Biological Chemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63100, the Departments of  Pharmacology and ** Biomolecular Chemistry, University of Wisconsin Medical School and the par  Program in Cellular and Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, and the Dagger Dagger  Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) utilize phosphatidylinositols containing D-3-position phosphates as substrates to form phosphatidylinositol 3,4-bisphosphate. In addition, type I PIP5Ks phosphorylate phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, while type II kinases have less activity toward this substrate. Remarkably, these kinases can convert phosphatidylinositol 3-phosphate to phosphatidylinositol 3,4,5-trisphosphate in a concerted reaction. Kinase activities toward the 3-position phosphoinositides are comparable with those seen with phosphatidylinositol 4-phosphate as the substrate. Therefore, the PIP5Ks can synthesize phosphatidylinositol 4,5-bisphosphate and two 3-phosphate-containing polyphosphoinositides. These unexpected activities position the PIP5Ks as potential participants in the generation of all polyphosphoinositide signaling molecules.


INTRODUCTION

Two distinct pathways have been characterized for agonist-stimulated signal transduction involving phosphatidylinositol (PtdIns).1 One pathway entails activation of phosphatidylinositol-specific phospholipase C by extracellular agonists resulting in the hydrolysis of phosphoinositides to generate soluble inositol phosphates including inositol 1,4,5-trisphosphate and diacylglycerol (reviewed in Refs. 1 and 2). The other pathway involves receptor-mediated activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) to produce the second messengers, phosphatidylinositol 3,4-bisphosphate (PtdIns 3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P3) (reviewed in Refs. 3 and 4).

A pathway for the formation of D-3-phosphatidylinositols, proposed based on kinetic studies of intact human neutrophils, is through phosphorylation of the D-3 position of the myo-inositol ring of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) by a PtdIns 4,5-P2 3-kinase and subsequent dephosphorylation of PtdIns 3,4,5-P3 to produce PtdIns 3,4-P2 (5). This pathway has been supported by the existence of the extensively characterized PtdIns 3-kinase enzyme family, which can catalyze in vitro phosphorylation of phosphatidylinositol 4-phosphate (PtdIns 4-P) and PtdIns 4,5-P2. Evidence for a different pathway for the formation of D-3 phosphatidylinositols has been found in human platelets, NIH 3T3 cells, and plants in which phosphorylation of the D-3-position of PtdIns to form PtdIns 3-P is followed by phosphorylation of the D-4-position to give PtdIns 3,4-P2 and then of the D-5-position to form PtdIns 3,4,5-P3 (6-10). The importance of these various routes of synthesis has been disputed. Indeed, until now, enzymes that catalyze the direct phosphorylation of PtdIns 3-P and PtdIns 3,4-P2 have not been clearly identified.

Several phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) have been discovered and characterized as enzymes synthesizing PtdIns 4,5-P2 (reviewed in Ref. 11). The best characterized isoforms are the type I and type II kinases that differ both biochemically and immunologically (12-14). The sequence of a type II PIP5K (PIP5KIIalpha ) has established that the PIP5K enzymes belong to a novel family (15). More recently, cDNAs encoding type I PIP5K (PIP5KI) and an additional PIP5KII isoform have been isolated (16-19). The translated sequences of PIP5KI and PIP5KII enzymes have only 35% amino acid identity in their kinase homology domains, further establishing the distinctiveness of these two subfamilies (16). The PIP5KIbeta cDNA is identical to a product of the gene reported to be mutated in Friedreich's ataxia, a common hereditary autosomal recessive disease (16, 20, 21). All four recombinant type I and II PIP5Ks have PtdIns 4-P 5-kinase activity (15-17, 19, 21).

None of the PIP5K isozymes have been examined for alternative substrates except phosphatidylinositol for which there was no detectable activity (12-14). We report here that the type I and II PIP5K isozymes also utilize the 3-phosphate-containing phosphatidylinositides, forming PtdIns 3,4-P2 and PtdIns 3,4,5-P3. This supports the existence of an additional pathway for the synthesis of 3-phosphate-containing phosphatidylinositol polyphosphates.


EXPERIMENTAL PROCEDURES

Materials

PtdIns and PtdIns 4-P were from Boehringer Mannheim or Sigma. PtdIns 3-P and PtdIns 3,4-P2 dipalmitoyl esters were synthesized (22) according to Chen and Prestwich,2 and Thum et al. (24), respectively. PtdIns 3-P and PtdIns 3,4-P2 dipalmitoyl esters were also from Matreya. [3H]inositol 1-phosphate, [3H]Ins 1,4-P2, [3H]Ins 1,3,4-P3 and [gamma -32P]ATP were from NEN Life Science Products. Lipofectamine, OPTI-MEM, and fetal bovine serum were obtained from Life Technologies, Inc. All other chemicals were purchased from Sigma.

Expression and Purification of Recombinant PIP5K Isozymes

Recombinant human PIP5Ks were expressed in Escherichia coli and purified by Ni2+-chelate chromatography (15, 16, 19). These proteins were stored in 50 mM Tris, pH 7.5, 1 mM EGTA, 150 mM NaCl, 0.01% sodium azide, and 20% glycerol for the type I isozymes or 50 mM Tris, pH 8.0, and 20% glycerol for the type II isozymes. Polymerase chain reaction-based cloning of the recombinant PIP5KIbeta used in these experiments resulted in two changes from the published sequence: glutamine 300 to arginine and serine 421 to proline. Both changes lie outside of the conserved kinase homology domain (16). The activity of PIP5KIIbeta is less stable; thus, larger amounts were used to achieve comparable activity in the kinase assays.

Phosphatidylinositol Phosphate Kinase Activity Assay

PIP5K isozymes were assayed in 50-µl reactions containing 50 mM Tris, pH 7.6, 10 mM MgCl2, 0.5 mM EGTA, 2-100 µM substrate prepared in isotonic KCl solution, and 50 µM ATP (4 µCi/nmol). The reactions, at 37 or 22 °C, ranged from 5 to 40 min. Immunoprecipitate kinase activities were tested for 10.5 min at 22 °C in 50 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 50 µM substrates prepared in Tris buffer and 50 µM ATP (4 µCi/nmol). The reactions were stopped by the addition of 100 µl of 1 N HCl, and the lipid products were extracted using 200 µl of chloroform/methanol (1:1). The organic phase was washed at least once in 80 µl of methanol, 1 N HCl (1:1). The organic phase was then spotted on TLC plates and run as before (16). After autoradiography for 1-12 h, the spots on the TLC plates corresponding to products were scraped into vials and counted using Cerenkov radioactivity in a Beckman scintillation counter.

Deacylation of Phosphatidylinositols and HPLC Analysis of Glycerophosphorylinositol Phosphates

Phosphatidylinositol products scraped from TLC plates were treated with methylamine reagent at 53 °C for 50 min (6). The glycerophosphorylinositol phosphate products of deacylation reactions were mixed with [3H]inositol 1-phosphate, [3H]Ins 1,4-P2, and [3H]Ins 1,3,4-P3 as internal standards and separated by HPLC with a Partisil 10 Sax column (Whatman) (25). The 1-ml fractions of eluate from the HPLC column were counted in a Beckman liquid scintillation counter.

Preparation of GroPIns [32P]3-P, GroPIns [32P]3,4-P2, and GroPIns [32P]3,4,5-P3 Standards

PtdIns [32P]3-P, PtdIns [32P]3,4-P2, and PtdIns [32P]3,4,5-P3 were prepared using a recombinant mutant p110 subunit of PtdIns 3-kinase that is constitutively active (26) (gift from L. T. Williams, Chiron). The Sf9 cell-expressed p110 mutant PtdIns 3-kinase was purified using the nickel-nitrolotriacetic acid affinity purification procedure (PharMingen). The kinase reactions were in 1 ml containing 20 mm Hepes, pH 7.5; 0.2 mM EDTA; 5 mM MgCl2; 1 mg of PtdIns, PtdIns 4-P, or PtdIns 4,5-P2; 1 mg of phosphatidylserine, 1 mCi of [gamma -32P]ATP, and 10 µg of PtdIns 3-kinase for 1 h at 37 °C. The lipid products were extracted and purified using preparative TLC (27). Purified PtdIns [32P]3-P, PtdIns [32P]3,4-P2, and PtdIns [32P]3,4,5-P3 were dried under N2 and deacylated (6) to produce glycerophosphorylinositol phosphate standards.

Preparation of Antibodies against Recombinant PIP5KIalpha

Rabbit polyclonal antibodies were raised against the hexahistine-PIP5KIalpha fusion protein (16) as before (13, 14). The antibodies were affinity-purified on the PIP5KIalpha fusion protein coupled to Sepharose, following established procedures (13, 14, 28). To serve as negative controls, preimmune IgGs and a depleted IgG pool were prepared using protein A-Sepharose (Sigma). The depleted IgG were those IgG isolated from immune sera after it had been depleted of anti-PIP5KIalpha antibodies by multiple cycles over the antigen affinity column. PIP5KII antibodies (13) were affinity-purified on a recombinant PIP5KIIalpha column (15). Western blots were performed as described previously (16).

Expression of PIP5Ks in COS-7 Cells

PIP5KIalpha and PIP5KIIalpha were N-terminally tagged with the FLAG epitope by subcloning their coding regions into pcDNA3-FLAG vectors provided by Dr. Jon Morrow (Yale University). The epitope is recognized by the anti-FLAG m2 monoclonal antibody (Eastman Kodak Co.). Plasmids were introduced into COS-7 cells by lipofectamine-mediated transfection following the manufacturer's procedures (Life Technologies, Inc.). Transfection of pcDNA3-CAT (Invitrogen) was used as a control. Transfected cells were harvested 17 h after adding the DNA and lysed in 250 µl of Nonidet P-40 lysis buffer.

Immunoprecipitation of PIP5Ks from COS-7 Cells

After they were rinsed with phosphate-buffered saline, COS-7 cells were lysed in 1.0% Nonidet P-40, 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin and 10 trypsin inhibitory units/ml aprotinin. The clarified lysates (250 µg) were precleared with Omnisorb cells (Calbiochem), the protein G matrix used to isolate the immune complexes. Immunoprecipitations were performed with 2 µg of antibody and the Omnisorb cells (28). For the transfection experiments, immunoprecipitations were done from 200 µl of lysate with 6 µg of alpha -FLAG antibody. After several washes, the pellets were resuspended in 80 µl of 50 mM Tris, pH 7.5, and split into 20-µl samples for kinase assays and Western blotting.


RESULTS

The ability of four different PIP5K isozymes to phosphorylate PtdIns 3-P, PtdIns 3,4-P2, and PtdIns 4-P is shown in Fig. 1A. When analyzed by TLC, the PtdIns 3-P phosphorylation product migrated as a phosphatidylinositol bisphosphate (PtdInsP2). The product migrated more slowly than PtdIns 4,5-P2, probably because the PtdIns 3-P substrate is a dipalmitoyl synthetic lipid (22). The two additional faint spots above PtdIns 3-P-derived PtdInsP2 may be phosphorylatable minor contaminants arising during the chemical synthesis of PtdIns 3-P. The PIP5KII enzymes have similar or greater activity toward PtdIns 3-P in comparison with PtdIns 4-P, whereas the PIP5KI isozymes prefer PtdIns 4-P. In addition, the PIP5KI enzymes also have activity toward PtdIns 3,4-P2, producing a product that migrates as PtdInsP3 (Fig. 1A, lanes 5 and 6). Remarkably, both PIP5KI isozymes and PIP5KIIalpha produce products from PtdIns 3-P that migrate in the PtdInsP3 position (Fig. 1A, lanes 1-3).


Fig. 1. Recombinant PIP5K isozymes phosphorylate 3-phosphate-containing phosphatidylinositols. A, the activities of E. coli-expressed recombinant PIP5K isozymes were assayed using 80 µM PtdIns 3-P, PtdIns 3,4-P2, or PtdIns 4-P for 10.5 min at 22 °C. The enzymes assayed were PIP5KIalpha (0.2 µg), PIP5KIbeta (0.7 µg), PIP5KIIalpha (2 µg), and PIP5KIIbeta (180 µg). The positions of products of the reaction are marked by arrows. All lanes were from the same TLC plate with different autoradiograph exposures. Exposures were for 5 min (lane 3) or 15 min (lanes 1, 2, and 9-11) at room temperature or 1.5 h at -80 °C (lanes 4-8 and 12). B, time course of PIP5KIIalpha activity. The kinase activity of PIP5KIIalpha (2.4 µg) toward 80 µM PtdIns 3-P (filled circles) or PtdIns 4-P (open squares) was assayed for 5-40 min at 37 °C. C, time course of PIP5KIalpha activity. The kinase activity of PIP5KIalpha (0.4 µg) toward 5 µM PtdIns 3-P (filled circles) or PtdIns 4-P (open squares) was determined for 0-20 min at 37 °C.
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The initial rate of PtdIns 3-P phosphorylation using PIP5KIIalpha was 4-fold greater than that of its previously identified substrate PtdIns 4-P (Fig. 1B). In addition, the phosphorylation of PtdIns 3-P was linear for a longer time interval compared with PtdIns 4-P (Fig. 1B). In a short reaction (5 min), comparison of PIP5KIIalpha kinase activity toward PtdIns 3-P and PtdIns 4-P at various concentrations indicated that the activity toward both substrates was dependent on their concentrations (data not shown). The kinetic parameters for PIP5KIIalpha phosphorylation of PtdIns 3-P and PtdIns 4-P are shown in Table I. The apparent Km value of PIP5KIIalpha for PtdIns 3-P is 3-fold greater than PtdIns 4-P. However, the Vm for PtdIns 3-P is also about 3-fold greater than that for PtdIns 4-P. As a result, the catalytic efficiency (Vm/Km) for these two substrates is the same. Little activity toward PtdIns 3,4-P2 was detected with the type II PIP5Ks (Fig. 1A, lanes 3 and 4).

Table I. Kinetic parameters of PIP5K isozymes

The data are representative of three separate sets of experiments. Units for kinetic values are µM for Km and pmol/min per mg of purified protein based on a Bradford assay for Vm.
Enzymes Substrates
PtdIns 3-P
PtdIns 4-P
PtdIns 3,4-P2
Km Vm Vm/Km Km Vm Vm/Km Km Vm Vm/Km

PIP5KIIalpha 120 90 0.8 50 39 0.8 NDa ND ND
PIP5KIalpha 65 1903 29.0 47 29653 631.0 80 438 5.5
PIP5KIbeta b 5 42 8.4 262 4753 18.0 6 39 6.5

a ND, none detected.
b Putative Friedrich's ataxia gene product.

The kinetic parameters of PIP5KI isozymes with their different substrates are listed in Table I. The time dependence of PIP5KIalpha activity using PtdIns 3-P and PtdIns 4-P is shown in Fig. 1C. The substrate preferences of the type I isozymes are different from the type II isozyme in that the type I PIP5K (alpha  and beta ) enzymes phosphorylate PtdIns 4-P with a much greater Vm than PtdIns 3-P. However, the Km values of the PIP5KI enzymes using PtdIns 3-P are lower than that of PIP5KIIalpha .

The products of the PIP5K reactions using PtdIns 3-P and PtdIns 3,4-P2 as substrates were identified by HPLC analysis (Fig. 2). It was anticipated that the PtdInsP2 products would be PtdIns 3,5-P2, given the specificity of the PIP5K enzymes toward PtdIns 4-P. Surprisingly, HPLC analysis demonstrated that the deacylated product of all of the PIP5K isozymes using PtdIns 3-P as substrate co-chromatographed with GroPIns 3,4-P2, as illustrated for PIP5KIIalpha in Fig. 2A. This indicated that these kinases synthesize PtdIns 3,4-P2. The HPLC analysis also revealed that the product of PIP5KIalpha and PIP5KIbeta activity toward PtdIns 3,4-P2 was PtdIns 3,4,5-P3. This is shown for PIP5KIalpha in Fig. 2B.


Fig. 2. HPLC analysis of the deacylated glycerophosphorylinositol products of PIP5K reactions. The deacylated products (open squares) of PIP5K reactions were analyzed using HPLC with [3H]Ins 1,4-P2 and [3H]Ins 1,3,4-P3 as internal standards. The lines with closed circles show the elution of [32P]GroPIns 3,4-P2 (A) and [32P]GroPIns 3,4,5-P3 (B) standards in parallel runs using the same internal standards. The elution of the internal standards marked by arrows was identical in each pair of runs. A, the deacylated PtdInsP2 product of PIP5KIIalpha using PtdIns 3-P as a substrate. B, the deacylated PtdInsP3 product of PIP5KIalpha using PtdIns 3,4-P2 as substrate. Different Partisil 10 Sax columns were used for A and B, so the elution positions of the internal standards were different.
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The structure of these products was further verified using specific inositol lipid phosphatases. The inositol polyphosphate 4-phosphatase (4-phosphatase) specifically hydrolyzes the 4-position phosphate of PtdIns 3,4-P2 (27). Treatment of the putative PtdIns 3,4-P2 products of PIP5K reactions with recombinant 4-phosphatase resulted in release of 32P-labeled inorganic phosphate, confirming that this was PtdIns 3,4-P2 labeled in the D-4-position (data not shown). In addition, treatment of the PtdInsP3 product with the Lowe oculocerebrorenal syndrome 5-phosphatase, an enzyme that specifically hydrolyzes the 5-position phosphate of PtdIns 3,4,5-P3 (29, 30), released 32P-labeled inorganic phosphate (data not shown). This result confirms that the product of this reaction was PtdIns 3,4,5-P3 labeled on the D-5-position.

Reactions using PtdIns 3-P as substrate also contained a product that migrated as PtdIns 3,4,5-P3. This product was observed using PIP5KI isozymes and PIP5KIIalpha but not PIP5KIIbeta (Fig. 1A, lanes 1-4). HPLC analysis confirmed that this was PtdIns 3,4,5-P3, which comigrated with lesser concentrations of lyso-PtdIns 3,4-P2 (20% for PIP5KIs, 45% for PIP5KIIalpha ). The amounts of PtdIns 3,4,5-P3 formed are shown in Table II. Because the substrate concentrations were 80 µM and the intermediate PtdIns 3,4-P2 product was nanomolar where PtdIns 3-P was the substrate, the amount of PtdIns 3,4,5-P3 formed is remarkable. Indeed, the amount of PtdIns 3,4,5-P3 formed from PtdIns 3-P using either PIP5KIalpha or PIP5KIIalpha was similar to that using 80 µM PtdIns 3,4-P2 with PIP5KIalpha . These results suggest that synthesis of PtdIns 3,4,5-P3 from PtdIns 3-P is a concerted reaction. In the case of the type II enzymes, PtdIns 3,4-P2 was not detectably phosphorylated by these enzymes. Yet, when type II kinases use PtdIns 3-P as substrate, PtdIns 3,4,5-P3 is produced.

Table II. Conversion of PtdIns 3-P to PtdIns 3,4,5-P3

Reactions were carried out in 50 µl, and the product concentration at the end of the reaction is listed. Reactions were for 10.5 min using 0.2 µg of PIP5KIalpha or 8 µg of PIP5KIIalpha . After TLC separation, the products were deacylated, and the glycerophosphorylinositol phosphate derivatives were determined by HPLC.
Isozyme Substrate (80 µM) PtdIns 3,4-P2 PtdIns 3,4,5-P3 Ratio of PtdIns 3,4,5-P3 to PtdIns 3,4-P2

µM µM × 104
PIP5KIalpha PtdIns 3-P 0.34 0.015 440
PtdIns 3,4-P2 0.02 2.5
PIP5KIIalpha PtdIns 3-P 0.65 0.0065 100
PtdIns 3,4-P2 NDa

a ND, none detected.

These data were obtained using recombinant, E. coli-expressed PIP5K isoforms, but similar results were observed using native PIP5Ks from mammalian cells. PIP5KII purified from erythrocytes had similar activity to the PIP5KIIalpha presented above (data not shown). The ability of PIP5KIalpha to phosphorylate the 3-phosphate-containing lipids was validated by immunoprecipitation of the kinase from COS-7 cells. When COS-7 cell lysates were Western blotted with anti-PIP5KIalpha antibody, a single 68-kDa protein was detected, which was immunoprecipitated with the same antibody (Fig. 3A). The PIP5KIalpha was not immunoprecipitated using an IgG depleted of PIP5KIalpha reactivity (Fig. 3A) or preimmune IgG (data not shown). The native PIP5KIalpha was able to phosphorylate both PtdIns 3-P and PtdIns 3,4-P2, and the activity toward the former was only 4-fold lower compared with PtdIns 4-P (Fig. 3B). In addition, the production of PtdIns 3,4,5-P3 from PtdIns 3-P was also observed.


Fig. 3. Substrate comparison of native and transiently transfected PIP5Ks immunoprecipitated from COS-7 cells. A, anti-PIP5KIalpha antibodies or control depleted IgG were used to immunoprecipitate from COS-7 cell lysates. Shown is a Western blot with anti-PIP5KIalpha antibody (2 µg/ml) of human red blood cell (hRBC) membranes, a COS-7 cell lysate, and the immunoprecipitates. B, the immunoprecipitates or recombinant PIP5KIalpha were assayed for activity toward 50 µM PtdIns 3-P, PtdIns 3,4-P2, or PtdIns 4-P for 10.5 min. The substrates are numbered based on the positions of the inositol ring already phosphorylated, i.e. 3 for PtdIns 3-P. The positions of the products of the reaction are marked by arrows. The autoradiograph exposures (same TLC plate) were 7 h for the immunoprecipitates and 4 h for the recombinant enzyme. C, the epitope-tagged PIP5KIalpha and PIP5KIIalpha were transfected into COS-7 cells and immunoprecipitated with the anti-FLAG m2 antibody. Transfection of untagged chloramphenicol acetyl transferase (CAT) served as a control for these experiments. The lysates of transfected cells and FLAG immunoprecipitates were transferred to an Immobilon-P membrane and Western blotted sequentially for PIP5KIalpha (2 µg/ml) and PIP5KIIalpha (10 µg/ml). The epitope-tagged kinases are slightly larger than the native enzymes. D, the anti-FLAG immunoprecipitates were assayed for kinase activity as before. A 5-h autoradiograph exposure is shown.
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The same pattern of phosphorylation of D3-phosphatidylinositols was observed using recombinant PIP5KIalpha and PIP5KIIalpha expressed in COS-7 cells (Fig. 3, C and D). PIP5KIalpha and PIP5KIIalpha containing a FLAG epitope at their N termini were transiently transfected into COS-7 cells, immunoprecipitated with an anti-FLAG monoclonal antibody, and assayed for activity using PtdIns 3-P, PtdIns 3,4-P2 and PtdIns 4-P. No PIP5K immunoreactivity (Fig. 3C) or activity (data not shown) was immunoprecipitated from the control transfected cells. The only difference between the PIP5Ks expressed in E. coli and mammalian cells was that the native and recombinant PIP5KIalpha expressed in COS cells had quantitatively greater activity toward PtdIns 3-P.


DISCUSSION

Here we report that the PIP5K isozymes are kinases with dual substrate specificity. They can phosphorylate PtdIns 4-P on the adjacent D-5-position. They can also phosphorylate 3-phosphate-containing phosphatidylinositols including PtdIns 3-P and PtdIns 3,4-P2 on the adjacent D-4- or D-5-positions. None of the other characterized phosphatidylinositol kinases are known to have this ability. The uniqueness of these enzymes is further emphasized by the lack of sequence homology with known phosphatidylinositol, inositol phosphate, or protein kinases (15, 16).

Our data demonstrate that the PIP5Ks have the in vivo potential to synthesize the signaling molecules PtdIns 3,4-P2 and PtdIns 3,4,5-P3, in addition to PtdIns 4,5-P2, as summarized in Fig. 4. This catalytic capacity was shown for both native and recombinant PIP5K isozymes. The production of both PtdIns 3,4-P2 and PtdIns 3,4,5-P3 when PtdIns 3-P is the initial substrate suggests that the product of the first reaction is retained on the enzyme and phosphorylated again in a concerted reaction. This result has important biological implications because it suggests an additional mechanism for PtdIns 3,4,5-P3 synthesis within cells.


Fig. 4. Phosphorylation of D-3-phosphate-containing phosphatidylinositols by PIP5Ks provides another route for PtdIns 3,4-P2 and PtdIns 3,4,5-P3 production. The multiple reactions that PIP5Ks catalyze are shown in the context of known phosphoinositide pathways leading to production of PtdIns 3,4,5-P3.
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There is evidence in the literature that a platelet PtdIns 3-P 4-kinase activity is stimulated by the thrombin receptor and protein kinase C activation (7, 8). PIP5KIIalpha is present in platelets (18, 31) and, as the only currently identified PtdIns 3-P 4-kinase in platelets, is a likely candidate for that PtdIns 3-P 4-kinase activity. Based on all of the available data, PIP5KIIalpha has properties indistinguishable from the platelet PtdIns 3-P 4-kinase (7, 8). PIP5KIIbeta is activated by its association with the TNF receptor (19) and could generate PtdIns 3,4-P2 involved in proliferation. The PIP5Ks could also be regulated by receptors that are known to stimulate PtdIns 3,4-P2 and PtdIns 3,4,5-P3 production upon agonist activation. As discussed previously, a pathway leading to synthesis of PtdIns 3,4-P2 and PtdIns 3,4,5-P3 has been proposed to occur by phosphorylation of PtdIns 4,5-P2 by an agonist-activated 3-kinase (5). However, these arguments were based upon the observation that this PtdIns 3-kinase will phosphorylate all phosphoinositides lacking a phosphate in the D-3-position and that PtdIns 3,4,5-P3 appears to be the initial product. With the data presented here, an alternative pathway in which PtdIns is phosphorylated by PtdIns 3-kinase and then phosphorylated by a PIP5K isoform is plausible, and the concerted reaction could explain why PtdIns 3,4,5-P3 appears first.

The PIP5Ks have the potential to produce three signaling molecules: PtdIns 4,5-P2, PtdIns 3,4-P2, and PtdIns 3,4,5-P3. It will be very interesting to determine if the substrate preferences of these PIP5K isozymes are altered by receptor activation or different regulators such as the small G-proteins Rac and Rho (23, 32, 33). It is conceivable that modulation of these activities will be both spatially and temporally regulated, and thus the PIP5K enzymes could participate in a plethora of cellular events by generating multiple messengers. Given the expanded substrate repertoire of these enzymes, we propose that they be designated as phosphatidylinositol phosphate 4/5-kinases.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants GM 51968 (to R. A. A.), HL 55672 (to P. W. M.), HL 16634 (to P. W. M.), and NS 29632 (to G. D. P.).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.
§   These authors contributed equally to this work.
§§   To whom correspondence should be addressed: Dept. of Pharmacology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-3753; Fax: 608-262-1257; E-mail: raanders{at}facstaff.wisc.edu.
1   The abbreviations used are: PtdIns, phosphatidylinositol; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; PIP5KI, type I phosphatidylinositol-4-phosphate 5-kinase; PIP5KII, type II phosphatidylinositol-4-phosphate 5-kinase; PtdIns 3-P, phosphatidylinositol 3-phosphate; PtdIns 4-P, phosphatidylinositol 4-phosphate; PtdIns 3,4-P2, phosphatidylinositol 3,4-bisphosphate; PtdIns 4,5-P2, phosphatidylinositol 4,5-bisphosphate; PtdInsP2, phosphatidylinositol bisphosphate; PtdIns 3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PtdInsP3, phosphatidylinositol trisphosphate; HPLC, high performance liquid chromatography; Ins 1,4-P2, inositol 1,4-bisphosphate; Ins 1,3,4-P3, inositol 1,3,4-trisphosphate; GroPIns, glycerophosphorylinositol.
2   J. Chen and G. D. Prestwich, submitted for publication.

ACKNOWLEDGEMENT

The pcDNA3-FLAG vectors were a gift of Dr. Jon Morrow (Yale University).


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