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

Open AccessPublished:July 11, 1997DOI:https://doi.org/10.1074/jbc.272.28.17756
      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.
      Two distinct pathways have been characterized for agonist-stimulated signal transduction involving phosphatidylinositol (PtdIns).
      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.
      1The 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.
      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.
      • Majerus P.W.
      and
      • Lee S.B.
      • Rhee S.G.
      ). 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.
      • Liscovitch M.
      • Cantley L.C.
      and
      • Divecha N.
      • Irvine R.F.
      ).
      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(
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ). 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 (
      • Cunningham T.W.
      • Lips D.L.
      • Bansal V.S.
      • Caldwell K.K.
      • Mitchell C.A.
      • Majerus P.W.
      ,
      • Yamamoto K.
      • Lapetina E.G.
      ,
      • Yamamoto K.
      • Graziani A.
      • Carpenter C.
      • Cantley L.C.
      • Lapetina E.G.
      ,
      • Cunningham T.W.
      • Majerus P.W.
      ,
      • Brearley C.A.
      • Hanke D.E.
      ). 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-P2have 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.
      • Loijens J.C.
      • Boronenkov I.V.
      • Parker G.J.
      • Anderson R.A.
      ). The best characterized isoforms are the type I and type II kinases that differ both biochemically and immunologically (
      • Ling L.E.
      • Schulz J.T.
      • Cantley L.C.
      ,
      • Bazenet C.E.
      • Ruano A.R.
      • Brockman J.L.
      • Anderson R.A.
      ,
      • Jenkins G.H.
      • Fisette P.L.
      • Anderson R.A.
      ). The sequence of a type II PIP5K (PIP5KIIα) has established that the PIP5K enzymes belong to a novel family (
      • Boronenkov I.V.
      • Anderson R.A.
      ). More recently, cDNAs encoding type I PIP5K (PIP5KI) and an additional PIP5KII isoform have been isolated (
      • Loijens J.C.
      • Anderson R.A.
      ,
      • Ishihara H.
      • Shibasaki Y.
      • Kizuki N.
      • Katagiri H.
      • Yazaki Y.
      • Asano T.
      • Oka Y.
      ,
      • Divecha N.
      • Truong O.
      • Hsuan J.J.
      • Hinchliffe K.A.
      • Irvine R.F.
      ,
      • Castellino A.M.
      • Parker G.J.
      • Boronenkov I.V.
      • Anderson R.A.
      • Chao M.V.
      ). 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 (
      • Loijens J.C.
      • Anderson R.A.
      ). The PIP5KIβ cDNA is identical to a product of the gene reported to be mutated in Friedreich's ataxia, a common hereditary autosomal recessive disease (
      • Loijens J.C.
      • Anderson R.A.
      ,
      • Carvajal J.J.
      • Pook M.A.
      • Doudney K.
      • Hillermann R.
      • Wilkes D.
      • al-Mahdawi S.
      • Williamson R.
      • Chamberlain S.
      ,
      • Carvajal J.J.
      • Pook M.A.
      • dos Santos M.
      • Doudney K.
      • Hillermann R.
      • Minogue S.
      • Williamson R.
      • Hsuan J.J.
      • Chamberlain S.
      ). All four recombinant type I and II PIP5Ks have PtdIns 4-P 5-kinase activity (
      • Boronenkov I.V.
      • Anderson R.A.
      ,
      • Loijens J.C.
      • Anderson R.A.
      ,
      • Ishihara H.
      • Shibasaki Y.
      • Kizuki N.
      • Katagiri H.
      • Yazaki Y.
      • Asano T.
      • Oka Y.
      ,
      • Castellino A.M.
      • Parker G.J.
      • Boronenkov I.V.
      • Anderson R.A.
      • Chao M.V.
      ,
      • Carvajal J.J.
      • Pook M.A.
      • dos Santos M.
      • Doudney K.
      • Hillermann R.
      • Minogue S.
      • Williamson R.
      • Hsuan J.J.
      • Chamberlain S.
      ).
      None of the PIP5K isozymes have been examined for alternative substrates except phosphatidylinositol for which there was no detectable activity (
      • Ling L.E.
      • Schulz J.T.
      • Cantley L.C.
      ,
      • Bazenet C.E.
      • Ruano A.R.
      • Brockman J.L.
      • Anderson R.A.
      ,
      • Jenkins G.H.
      • Fisette P.L.
      • Anderson R.A.
      ). 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.

      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.1 A. 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 (
      • Prestwich G.D.
      ). 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. 1 A, lanes 5 and6). Remarkably, both PIP5KI isozymes and PIP5KIIα produce products from PtdIns 3-P that migrate in the PtdInsP3position (Fig. 1 A, lanes 1–3).
      Figure thumbnail gr1
      Figure 1Recombinant 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 PIP5KIα (0.2 μg), PIP5KIβ (0.7 μg), PIP5KIIα (2 μg), and PIP5KIIβ (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 PIP5KIIα activity. The kinase activity of PIP5KIIα (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 PIP5KIα activity. The kinase activity of PIP5KIα (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.
      The initial rate of PtdIns 3-P phosphorylation using PIP5KIIα was 4-fold greater than that of its previously identified substrate PtdIns 4-P (Fig. 1 B). In addition, the phosphorylation of PtdIns 3-P was linear for a longer time interval compared with PtdIns 4-P (Fig. 1 B). In a short reaction (5 min), comparison of PIP5KIIα 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 PIP5KIIα phosphorylation of PtdIns 3-P and PtdIns 4-P are shown in Table I. The apparent K m value of PIP5KIIα for PtdIns 3-P is 3-fold greater than PtdIns 4-P. However, the V m for PtdIns 3-P is also about 3-fold greater than that for PtdIns 4-P. As a result, the catalytic efficiency (V m /K m ) for these two substrates is the same. Little activity toward PtdIns 3,4-P2 was detected with the type II PIP5Ks (Fig. 1 A, lanes 3 and 4).
      Table IKinetic parameters of PIP5K isozymes
      EnzymesSubstrates
      PtdIns 3-PPtdIns 4-PPtdIns 3,4-P2
      K mV mV m /K mK mV mV m /K mK mV mV m /K m
      PIP5KIIα120900.850390.8ND
      ND, none detected.
      NDND
      PIP5KIα65190329.04729653631.0804385.5
      PIP5KIβ
      Putative Friedrich's ataxia gene product.
      5428.4262475318.06396.5
      The data are representative of three separate sets of experiments. Units for kinetic values are μm for K m and pmol/min per mg of purified protein based on a Bradford assay forV m .
      1-a ND, none detected.
      1-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 PIP5KIα activity using PtdIns 3-P and PtdIns 4-P is shown in Fig.1 C. The substrate preferences of the type I isozymes are different from the type II isozyme in that the type I PIP5K (α and β) enzymes phosphorylate PtdIns 4-P with a much greaterV m than PtdIns 3-P. However, theK m values of the PIP5KI enzymes using PtdIns 3-P are lower than that of PIP5KIIα.
      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 PtdInsP2products 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 PIP5KIIα in Fig.2 A. This indicated that these kinases synthesize PtdIns 3,4-P2. The HPLC analysis also revealed that the product of PIP5KIα and PIP5KIβ activity toward PtdIns 3,4-P2 was PtdIns 3,4,5-P3. This is shown for PIP5KIα in Fig.2 B.
      Figure thumbnail gr2
      Figure 2HPLC 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. Thelines 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 PIP5KIIα using PtdIns 3-P as a substrate. B, the deacylated PtdInsP3 product of PIP5KIα 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.
      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 (
      • Norris F.A.
      • Majerus P.W.
      ). 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 (
      • Zhang X.
      • Jefferson A.B.
      • Auethavekiat V.A.
      • Majerus P.W.
      ,
      • Jefferson A.B.
      • Auethavekiat V.
      • Pot D.A.
      • Williams L.T.
      • Majerus P.W.
      ), released32P-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 PIP5KIIα but not PIP5KIIβ (Fig.1 A, 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 PIP5KIIα). 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 PIP5KIα or PIP5KIIα was similar to that using 80 μm PtdIns 3,4-P2 with PIP5KIα. 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 IIConversion of PtdIns 3-P to PtdIns 3,4,5-P 3
      IsozymeSubstrate (80 μm)PtdIns 3,4-P2PtdIns 3,4,5-P3Ratio of PtdIns 3,4,5-P3 to PtdIns 3,4-P2
      μmμm× 10 4
      PIP5KIαPtdIns 3-P0.340.015440
      PtdIns 3,4-P20.022.5
      PIP5KIIαPtdIns 3-P0.650.0065100
      PtdIns 3,4-P2ND
      ND, none detected.
      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 PIP5KIα or 8 μg of PIP5KIIα. After TLC separation, the products were deacylated, and the glycerophosphorylinositol phosphate derivatives were determined by HPLC.
      2-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 PIP5KIIα presented above (data not shown). The ability of PIP5KIα 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-PIP5KIα antibody, a single 68-kDa protein was detected, which was immunoprecipitated with the same antibody (Fig.3 A). The PIP5KIα was not immunoprecipitated using an IgG depleted of PIP5KIα reactivity (Fig. 3 A) or preimmune IgG (data not shown). The native PIP5KIα 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. 3 B). In addition, the production of PtdIns 3,4,5-P3 from PtdIns 3-P was also observed.
      Figure thumbnail gr3
      Figure 3Substrate comparison of native and transiently transfected PIP5Ks immunoprecipitated from COS-7 cells. A, anti-PIP5KIα antibodies or control depleted IgG were used to immunoprecipitate from COS-7 cell lysates. Shown is a Western blot with anti-PIP5KIα antibody (2 μg/ml) of human red blood cell (hRBC) membranes, a COS-7 cell lysate, and the immunoprecipitates. B, the immunoprecipitates or recombinant PIP5KIα 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 PIP5KIα and PIP5KIIα 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 PIP5KIα (2 μg/ml) and PIP5KIIα (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.
      The same pattern of phosphorylation of D3-phosphatidylinositols was observed using recombinant PIP5KIα and PIP5KIIα expressed in COS-7 cells (Fig. 3, C and D). PIP5KIα and PIP5KIIα 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.3 C) 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 PIP5KIα 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 (
      • Boronenkov I.V.
      • Anderson R.A.
      ,
      • Loijens J.C.
      • Anderson R.A.
      ).
      Our data demonstrate that the PIP5Ks have the in vivopotential 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.
      Figure thumbnail gr4
      Figure 4Phosphorylation 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.
      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 (
      • Yamamoto K.
      • Lapetina E.G.
      ,
      • Yamamoto K.
      • Graziani A.
      • Carpenter C.
      • Cantley L.C.
      • Lapetina E.G.
      ). PIP5KIIα is present in platelets (
      • Divecha N.
      • Truong O.
      • Hsuan J.J.
      • Hinchliffe K.A.
      • Irvine R.F.
      ,
      • Hinchliffe K.A.
      • Irvine R.F.
      • Divecha N.
      ) 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, PIP5KIIα has properties indistinguishable from the platelet PtdIns 3-P 4-kinase (
      • Yamamoto K.
      • Lapetina E.G.
      ,
      • Yamamoto K.
      • Graziani A.
      • Carpenter C.
      • Cantley L.C.
      • Lapetina E.G.
      ). PIP5KIIβ is activated by its association with the TNF receptor (
      • Castellino A.M.
      • Parker G.J.
      • Boronenkov I.V.
      • Anderson R.A.
      • Chao M.V.
      ) 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-P3has been proposed to occur by phosphorylation of PtdIns 4,5-P2 by an agonist-activated 3-kinase (
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ). 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 (
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ,
      • Chong L.D.
      • Traynor-Kaplan A.
      • Bokoch G.M.
      • Schwartz M.A.
      ,
      • Ren X.-D.
      • Bokoch G.M.
      • Traynor-Kaplan A.
      • Jenkins G.H.
      • Anderson R.A.
      • Schwartz M.A.
      ). 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.

      Acknowledgments

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

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