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Phosphatidylinositol (3,4,5)P3 Is Essential but Not Sufficient for Protein Kinase B (PKB) Activation; Phosphatidylinositol (3,4)P2 Is Required for PKB Phosphorylation at Ser-473

STUDIES USING CELLS FROM SH2-CONTAINING INOSITOL-5-PHOSPHATASE KNOCKOUT MICE*
Open AccessPublished:January 07, 2002DOI:https://doi.org/10.1074/jbc.M106755200
      Using bone marrow derived mast cells from SH2-containing inositol-5-phosphatase (SHIP) +/+ and −/− mice, we found that the loss of SHIP leads to a dramatic increase in Steel Factor (SF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), a substantial reduction in PI(3,4)P2, and no change in PI(4,5)P2 levels. We also found that SF-induced activation of protein kinase B (PKB) is increased and prolonged in SHIP−/− cells, due in large part to more PKB associating with the plasma membrane in these cells. Pretreatment of SHIP−/− cells with 25 μm LY294002 resulted in complete inhibition of SF-induced PI(3,4)P2, while still yielding PI(3,4,5)P3 levels similar to those achieved in SHIP+/+ cells. This offered a unique opportunity to study the regulation of PKB by PI(3,4,5)P3, in the absence of PI(3,4)P2. Under these conditions, PKB activity was markedly reduced compared with that in SF-stimulated SHIP+/+ cells, even though more PKB localized to the plasma membrane. Although phosphoinositide-dependent kinase 1 mediated phosphorylation of PKB at Thr-308 was unaffected by LY294002, phosphorylation at Ser-473 was dramatically reduced. Moreover, intracellular delivery of PI(3,4)P2 to LY294002-pretreated, SF-stimulated SHIP−/− cells increased phosphorylation of PKB at Ser-473 and increased PKB activity. These results are consistent with a model in which SHIP serves as a regulator of both activity and subcellular localization of PKB.
      SH2
      src homology 2
      BMMCs
      bone marrow-derived mast cells
      PDK1
      phosphoinositide-dependent kinase 1
      PI3K
      phosphatidylinositol 3-kinase
      PI4K
      phosphatidylinositol 4-kinase
      PKB
      protein kinase B
      PMSF
      phenylmethylsulfonyl fluoride
      SF
      Steel factor or stem cell factor
      SHIP
      SH2-containing inositol-5-phosphatase
      PI(4)P
      phosphatidylinositol 4-phosphate
      PI(3
      4)P2, phosphatidylinositol 3,4-bisphosphate
      PI(4
      5)P2, phosphatidylinositol 4,5-bisphosphate
      PI(3
      4,5)P3, phosphatidylinositol 3,4,5-trisphosphate
      FCS
      fetal calf serum
      HPLC
      high performance liquid chromatography
      PH
      pleckstrin homology
      ILK
      integrin-linked kinase
      IMDM
      Iscove's modified Dulbecco's medium
      PTEN
      phosphatase and tensin homolog on chromosome 10
      di-C8
      dicapryloyl
      di-C16
      dipalmitoyl
      The src homology 2 (SH21)-containing inositol phosphatase (SHIP) is a 145-kDa hemopoietic-specific signaling protein (
      • Damen J.E.
      • Liu L.
      • Rosten P.
      • Humphries R.K.
      • Jefferson A.B.
      • Majerus P.W.
      • Krystal G.
      ,
      • Kavanaugh W.M.
      • Pot D.A.
      • Chin S.M.
      • Deuter-Reinhard M.
      • Jefferson A.B.
      • Norris F.A.
      • Masiarz F.R.
      • Cousens L.S.
      • Majerus P.W.
      • Williams L.T.
      ,
      • Lioubin M.N.
      • Algate P.A.
      • Tsai S.
      • Carlberg K.
      • Aebersold A.
      • Rohrschneider L.R.
      ) that becomes both tyrosine-phosphorylated and associated with the adapter protein Shc in response to many cytokines and to B and T cell receptor engagement (
      • Liu L.
      • Damen J.E.
      • Ware M.
      • Hughes M.
      • Krystal G.
      ). SHIP has been shown to inhibit immune receptor activation in both mast cells and B cells by binding to the tyrosine-phosphorylated immunoreceptor tyrosine-based inhibition motif of the inhibitory co-receptor FcγRIIB and inhibiting FcεR1- and B cell receptor-induced calcium influx, respectively (
      • Ono M.
      • Bolland S.
      • Tempst P.
      • Ravetch J.V.
      ,
      • Ono M.
      • Okada H.
      • Bolland S.
      • Yanagi S.
      • Kurosaki T.
      • Ravetch J.V.
      ). In addition, SHIP has been shown, even in the absence of FcγRIIB co-clustering, to play a “gatekeeper” role in IgE-mediated mast cell degranulation by setting the threshold for and limiting the degranulation process (
      • Huber M.
      • Helgason C.D.
      • Scheid M.P.
      • Duronio V.
      • Humphries R.K.
      • Krystal G.
      ,
      • Huber M.
      • Helgason C.D.
      • Damen J.E.
      • Liu L.
      • Humphries R.K.
      • Krystal G.
      ).
      In 1996 when we and others first reported the cloning of SHIP (
      • Damen J.E.
      • Liu L.
      • Rosten P.
      • Humphries R.K.
      • Jefferson A.B.
      • Majerus P.W.
      • Krystal G.
      ,
      • Kavanaugh W.M.
      • Pot D.A.
      • Chin S.M.
      • Deuter-Reinhard M.
      • Jefferson A.B.
      • Norris F.A.
      • Masiarz F.R.
      • Cousens L.S.
      • Majerus P.W.
      • Williams L.T.
      ,
      • Lioubin M.N.
      • Algate P.A.
      • Tsai S.
      • Carlberg K.
      • Aebersold A.
      • Rohrschneider L.R.
      ), we demonstrated its ability, in vitro, to hydrolyze the 5′-phosphate from phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) but not from PI(4,5)P2. More recently, however, by modifying the in vitro assay conditions, SHIP was found capable of readily hydrolyzing PI(4,5)P2 to PI(4)P (
      • Kisseleva M.V.
      • Wilson M.P.
      • Majerus P.W.
      ,
      • Rameh L.E.
      • Tolias K.F.
      • Duckworth B.C.
      • Cantley L.C.
      ). To resolve its phospholipid substrate specificity and to gain some insight into the normal role that SHIP plays in vivo, we generated a SHIP knockout mouse by homologous recombination in embryonic stem cells (
      • Helgason C.D.
      • Damen J.E.
      • Rosten P.
      • Grewal R.
      • Sorensen P.
      • Chappel S.M.
      • Borowski A.
      • Jirik F.
      • Krystal G.
      • Humphries R.K.
      ). Although these mice are viable and fertile, they suffer from progressive splenomegaly, massive myeloid infiltration of the lungs, wasting, and a shortened lifespan (
      • Helgason C.D.
      • Damen J.E.
      • Rosten P.
      • Grewal R.
      • Sorensen P.
      • Chappel S.M.
      • Borowski A.
      • Jirik F.
      • Krystal G.
      • Humphries R.K.
      ,
      • Liu Q.
      • Sasaki T.
      • Kozieradzki I.
      • Wakeham A.
      • Itie A.
      • Dumont D.J.
      • Penninger J.M.
      ). Interestingly, granulocyte/macrophage progenitors from these mice are substantially more responsive to multiple cytokines than their wild type littermates (
      • Helgason C.D.
      • Damen J.E.
      • Rosten P.
      • Grewal R.
      • Sorensen P.
      • Chappel S.M.
      • Borowski A.
      • Jirik F.
      • Krystal G.
      • Humphries R.K.
      ,
      • Liu Q.
      • Sasaki T.
      • Kozieradzki I.
      • Wakeham A.
      • Itie A.
      • Dumont D.J.
      • Penninger J.M.
      ). These mice have allowed us to ask whether one of SHIP's normal functions is to hydrolyze PI(3,4,5)P3 and/or PI(4,5)P2 in vivo. Specifically, in the present study we have utilized bone marrow-derived mast cells (BMMCs) from SHIP−/− and +/+ littermates to determine if SHIP affects PI(4,5)P2 levels and if it plays a significant role in hydrolyzing the Steel Factor (SF)-stimulated increase in PI(3,4,5)P3 in vivo. We have also used these two cell types to examine the role of SHIP in the activation of the proto-oncogene, protein kinase B (PKB) (also referred to as Akt or RAC) (
      • Alessi D.R.
      • Cohen P.
      ), and more specifically, the role of PI(3,4,5)P3 and PI(3,4)P2 in mediating the activation of PKB. Our data reveal that SHIP is the primary enzyme responsible for hydrolyzing PI(3,4,5)P3 in SF-stimulated normal BMMCs and that PI(3,4,5)P3 is the major source for PI(3,4)P2 in these cells. However, the presence or absence of SHIP does not have any significant effect on PI(4,5)P2levels. Interestingly, although the loss of SHIP increases the levels of PI(3,4,5)P3 and enhances PKB activity, we show that the generation of PI(3,4)P2 is also essential to fully activate PKB, because this lipid is important for mediating phosphorylation of PKB at Ser-473.

      DISCUSSION

      The current model for the activation of PKB suggests that inactive, cytosolic PKB translocates to the plasma membrane to bind to PI(3,4,5)P3 and/or PI(3,4)P2 that is generated in response to PI3K activation (
      • Toker A.
      • Cantley L.C.
      ,
      • Hemmings B.A.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Downward J.
      ). Binding via its pleckstrin homology (PH) domain to one or both of these phosphoinositides alters PKB's conformation such that it becomes accessible to phosphorylation by two upstream kinases, PDK1 and an unidentified kinase that has been referred to as PDK2. The phosphatidylinositol-dependent kinase PDK1 (
      • Alessi D.R.
      • Deak M.
      • Casamayor A.
      • Caudwell F.B.
      • Morrice N.
      • Norman D.G.
      • Gaffney P.
      • Reese C.B.
      • MacDougall C.N.
      • Harbison D.
      • Ashworth A.
      • Bownes M.
      ,
      • Stephens L.
      • Anderson K.
      • Stokoe D.
      • Erdjument-Bromage H.
      • Painter G.F.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • McCormick F.
      • Tempst P.
      • Coadwell J
      • Hawkins P.T.
      ,
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.J.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ) phosphorylates PKB in the activation loop of its kinase domain at Thr-308 while a separate kinase phosphorylates PKB in its carboxyl-terminal tail at Ser-473. A recent report has described this as Hm kinase due to its phosphorylation at the hydrophobic region of PKB (
      • Biondi R.M.
      • Kieloch A.
      • Currie R.A.
      • Deak M.
      • Alessi D.R.
      ). Another possible route for control of Ser-473 phosphorylation could be through autophosphorylation, as suggested by Toker and Newton (
      • Toker A.
      • Newton A.C.
      ). However, Ser-473 phosphorylation occurs normally in PDK-1-deficient ES cells, which lack Thr-308 phosphorylation and PKB activity (
      • Williams M.R.
      • Arthur J.S.
      • Balendran A.
      • van der Kaay J.
      • Poli V.
      • Cohen P.
      • Alessi D.R.
      ). Furthermore, a recent study showed that staurosporine can uncouple PKB activity from Ser-473 phosphorylation (
      • Hill M.M.
      • Andjelkovic M.
      • Brazil D.P.
      • Ferrari S.
      • Fabbro D.
      • Hemmings B.A.
      ). Thus, PKB autophosphorylation is not likely to be the mechanism by which Ser-473 is regulated. Once phosphorylated at both sites, PKB becomes locked into the fully active conformation, detaches from the plasma membrane, phosphorylates target substrates such as glycogen synthase kinase-3 (
      • Cross D.A.E.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ), caspase 9 (
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ), forkhead transcription factors (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ,
      • Kops G.J.P.L.
      • De de Ruiter N.D.
      • Vries-Smits A.M.
      • Powell D.R.
      • Bos J.L.
      • Burgering B.M.
      ), IKKα (
      • Romashkova J.A.
      • Makarov S.S.
      ), eNOS (
      • Dimmeler S.
      • Fleming I.
      • Fisslthaler B.
      • Hermann C.
      • Busse R.
      • Zeiher A.M.
      ,
      • Fulton D.
      • Gratton J.P.
      • McCabe T.J.
      • Fontana J.
      • Fujio Y.
      • Walsh K.
      • Franke T.F.
      • Papapetropoulos A.
      • Sessa W.C.
      ), and Raf (
      • Zimmermann S.
      • Moelling K.
      ), and it also translocates into the nucleus (
      • Andjelkovic M.
      • Alessi D.R.
      • Meier R.
      • Fernandez A.
      • Lamb N.J.
      • Frech M.
      • Cron P.
      • Cohen P.
      • Lucocq J.M.
      • Hemmings B.A.
      ).
      One major controversy surrounding this model of PKB activation is whether PI(3,4,5)P3 or PI(3,4)P2 is the critical second messenger that attracts PKB and its kinases to the plasma membrane in vivo. There is substantial in vitro data suggesting that PI(3,4)P2 has a higher affinity than PI(3,4,5)P3 for PKB (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Frech M.
      • Andjelkovic M.
      • Ingley E.
      • Reddy K.K.
      • Falck J.R.
      • Hemmings B.A.
      ,
      • Klippel A.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ). Moreover, lipid vesicles containing PI(3,4)P2 have been shown to modestly activate PKB, perhaps via dimerization (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Frech M.
      • Andjelkovic M.
      • Ingley E.
      • Reddy K.K.
      • Falck J.R.
      • Hemmings B.A.
      ,
      • Klippel A.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ), whereas vesicles containing PI(3,4,5)P3 have been reported to either inhibit (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Frech M.
      • Andjelkovic M.
      • Ingley E.
      • Reddy K.K.
      • Falck J.R.
      • Hemmings B.A.
      ) or have no effect (
      • Klippel A.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ). As well, in vivo data indicate that addition of di-C16-PI(3,4)P2 to serum-starved NIH 3T3 cells stimulates PKB autophosphorylation whereas di-C16-PI(3,4,5)P3 causes slight inhibition (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ). Finally, platelet studies have shown that PKB activation correlates with PI(3,4)P2 rather than PI(3,4,5)P3 production following thrombin stimulation (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ), and integrin cross-linking has been reported to generate PI(3,4)P2 but not PI(3,4,5)P3 and results in PKB activation (
      • Banfic H.
      • Downes C.P.
      • Rittenhouse S.E.
      ).
      However, in support of PI(3,4,5)P3 being the critical second messenger, it also binds PKB and in one study examining the binding of di-C8-PI(3,4,5)P3 and PI(3,4)P2 to purified PKB, PI(3,4,5)P3 bound with slightly higher affinity (
      • Frech M.
      • Andjelkovic M.
      • Ingley E.
      • Reddy K.K.
      • Falck J.R.
      • Hemmings B.A.
      ). Other studies have shown that PDK1, which also possesses a PH domain, has a substantially higher affinity for PI(3,4,5)P3 than for PI(3,4)P2 (
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.J.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ) and needs to interact with the plasma membrane to phosphorylate PKB efficiently (
      • Anderson K.E.
      • Coadwell J.
      • Stephens L.R.
      • Hawkins P.T.
      ). In fact, Alessi et al. (
      • Alessi D.R.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.A.
      ) have proposed that PKB activation by PI(3,4)P2 in vitro might be due to contamination of the PKB preparations with PDK1. Last, phosphorylation of PKB at Ser-473 also appears to be under the control of PI3K (
      • Alessi D.R.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.A.
      ), but because the kinase has not as yet been identified, there is no data to suggest whether it may have a higher affinity for PI(3,4,5)P3 or PI(3,4)P2.
      The availability of SHIP−/− BMMCs has given us the unique opportunity to modulate these two phosphoinositides, such that in the presence of 25 μm LY294002 an amount of PI(3,4,5)P3equivalent to that observed in SHIP+/+ BMMCs is produced in response to SF (Fig. 5 A). Under these unique conditions, no PI(3,4)P2 is formed and we were therefore able to investigate the activation of PKB in vivo, in the presence of increased PI(3,4,5)P3 alone. Comparison of PKB levels in the membrane fraction of SF-stimulated SHIP+/+ cells, with LY294002-pretreated SHIP−/− cells, suggests that PI(3,4,5)P3 alone is sufficient in vivo to attract PKB to the plasma membrane. However, our experiments also demonstrated that membrane recruitment via PI(3,4,5)P3alone is not sufficient to drive Ser-473 phosphorylation, which was prevented by LY294002 pretreatment under these conditions. This led us to consider the possibility that the generation of PI(3,4)P2 is necessary for Ser-473 phosphorylation. To test this, we added exogenous PI(3,4)P2, which restored both Ser-473 phosphorylation and activity. These data, and our results with two structurally unrelated PI3K inhibitors, strongly support the possibility that the Ser-473 phosphorylation of PKB is dependent upon the presence of PI(3,4)P2.
      It is also important to note that PDK1-mediated phosphorylation of PKB at Thr-308 in SHIP−/− BMMCs is unaffected by 25 μmLY294002 (Fig. 6 B), suggesting that PI(3,4,5)P3alone is sufficient to impart the conformational change within PKB to allow its phosphorylation by PDK1. In addition, our results demonstrate that little or no phosphorylation of Ser-473 is required to dock PDK-1 and mediate phosphorylation of Thr-308. This could be in contrast to other AGC kinases, such as RSK2, in which phosphorylation of the Ser-473-equivalent residue is thought to provide a docking site for PDK-1 to allow efficient activation loop phosphorylation (
      • Frodin M.
      • Jensen C.J.
      • Merienne K.
      • Gammeltoft S.
      ).
      We thus propose a model of SF-stimulated PKB activation in BMMCs in which PKB and PDK1 are attracted via PI(3,4,5)P3 whereas a Ser-473 kinase is attracted via PI(3,4)P2 to the plasma membrane (Fig. 9). Another possibility could be that PI(3,4)P2 inactivates a phosphatase responsible for the turnover of Ser-473 phosphorylation, and at this point we cannot rule out this possibility. Hemmings and co-workers (
      • Andjelkovic M.
      • Maira S.M.
      • Cron P.
      • Parker P.J.
      • Hemmings B.A.
      ) have used a membrane-inducible PKB allele to suggest that Ser-473 phosphorylation could be under the control of a PI3K-dependent phosphatase activity, but the mechanism for this is currently unknown. The same group found that hyperosmotic stress caused dephosphorylation of PKB at Ser-473 while also causing elevation of PI(3,4,5)P3 levels and depression of PI(3,4)P2, thus likely affecting 5-phosphatase activity (
      • Meier R.
      • Thelen M.
      • Hemmings B.A.
      ). PI(3,4)P2 inhibition of a Ser-473 phosphatase could also allow for the possibility that this residue is a target of autophosphorylation by PKB. In either case, our data show that generation of PI(3,4)P2 leads to accumulation of Ser-473 phosphorylation. Of note, it has been proposed that, because PDK1 has such a high affinity for PI(3,4,5)P3 (
      • Stephens L.
      • Anderson K.
      • Stokoe D.
      • Erdjument-Bromage H.
      • Painter G.F.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • McCormick F.
      • Tempst P.
      • Coadwell J
      • Hawkins P.T.
      ), there might be sufficient PI(3,4,5)P3 at the plasma membrane in unstimulated cells to localize significant levels of this enzyme (
      • Anderson K.E.
      • Coadwell J.
      • Stephens L.R.
      • Hawkins P.T.
      ). It is therefore possible that there are only two rate-limiting steps in PKB activation: PKB localization and PI(3,4)P2-mediated phosphorylation of Ser-473.
      Figure thumbnail gr9
      Figure 9Model of SF-stimulated PKB activation in BMMCs. Details of the model are discussed in the text. Our results support a role for PI(3,4)P2, generated by SHIP activity, in the activation of PKB by promoting phosphorylation at Ser-473.
      An issue that remains to be resolved is whether the integrin-linked kinase (ILK) may be the Ser-473 kinase. Delcommenne et al.(
      • Delcommenne M.
      • Tan C.
      • Gray V.
      • Rue L.
      • Woodgett J.
      • Dedhar S.
      ) have reported that ILK may act as the putative PDK2. This protein has a PH-like domain that appears to be capable, at least in vitro, of binding to both PI(3,4,5)P3 and, to a lesser extent, PI(3,4)P2. More recent data further supports the role of ILK as a direct regulator of PKB activity (
      • Persad S.
      • Attwell S.
      • Gray V.
      • Mawji N.
      • Deng J.T.
      • Leung D.
      • Yan J.
      • Sanghera J.
      • Walsh M.P.
      • Dedhar S.
      ). Until some insight into the in vivo affinities of ILK for PI(3,4)P2 is obtained, our present findings cannot help in determining whether ILK is serving as the kinase that phosphorylates Ser-473 in BMMCs.
      Interestingly, with regards to SHIP, our studies to date as well as others have shown that this inositol-5-phosphatase plays a negative role in proliferation (
      • Lioubin M.N.
      • Algate P.A.
      • Tsai S.
      • Carlberg K.
      • Aebersold A.
      • Rohrschneider L.R.
      ,
      • Liu L.
      • Damen J.E.
      • Ware M.
      • Hughes M.
      • Krystal G.
      ,
      • Helgason C.D.
      • Damen J.E.
      • Rosten P.
      • Grewal R.
      • Sorensen P.
      • Chappel S.M.
      • Borowski A.
      • Jirik F.
      • Krystal G.
      • Humphries R.K.
      ), survival (
      • Liu Q.
      • Sasaki T.
      • Kozieradzki I.
      • Wakeham A.
      • Itie A.
      • Dumont D.J.
      • Penninger J.M.
      ,
      • Liu L.
      • Damen J.E.
      • Hughes M.R.
      • Babic I.
      • Jirik F.R.
      • Krystal G.
      ), and end cell activation (
      • Ono M.
      • Bolland S.
      • Tempst P.
      • Ravetch J.V.
      ,
      • Ono M.
      • Okada H.
      • Bolland S.
      • Yanagi S.
      • Kurosaki T.
      • Ravetch J.V.
      ,
      • Huber M.
      • Helgason C.D.
      • Scheid M.P.
      • Duronio V.
      • Humphries R.K.
      • Krystal G.
      ,
      • Huber M.
      • Helgason C.D.
      • Damen J.E.
      • Liu L.
      • Humphries R.K.
      • Krystal G.
      ). However, the data presented herein suggest that SHIP may also play a positive role by generating PI(3,4)P2 and thus activating PKB. This raises several interesting points about the regulation of PKB. It seems that nature has set in place the requirement of both PI(3,4,5)P3 and PI(3,4)P2for its activation. Loss of SHIP may not super-potentiate PKB activity, because PI(3,4)P2 levels are reduced, and this may explain why we have not observed any hemopoietic malignancies in mice lacking this hemopoietically expressed gene (
      • Helgason C.D.
      • Damen J.E.
      • Rosten P.
      • Grewal R.
      • Sorensen P.
      • Chappel S.M.
      • Borowski A.
      • Jirik F.
      • Krystal G.
      • Humphries R.K.
      ). However, in mice heterozygous for the inositol-3-phosphatase, PTEN, many tumors, including lymphomas, appear (
      • Podsypanina K.
      • Ellenson L.H.
      • Nemes A.
      • Gu J.
      • Tamura M.
      • Yamada K.M.
      • Cordon-Cardo C.
      • Catoretti G.
      • Fisher P.E.
      • Parsons R.
      ). Because a reduction in PTEN reduces the hydrolysis of PI(3,4,5)P3 back to PI(4,5)P2, it allows for an increase in both PI(3,4,5)P3 and PI(3,4)P2 (
      • Stambolic V.
      • Suzuki A.
      • de la Pompa J.L.
      • Brothers G.M.
      • Mirtsos C.
      • Sasaki T.
      • Ruland J.
      • Penninger J.M.
      • Siderovski D.P.
      • Mak T.W.
      ) and perhaps greater PKB activity than can be obtained in SHIP-depleted cells, and this may facilitate tumor formation. Studies are currently underway to directly compare PKB activity in response to cytokine stimulation of SHIP−/− and PTEN−/− ES cell-derived mast cells to assess this possibility.
      In summary, we have shown that SHIP is the primary enzyme responsible for hydrolyzing PI(3,4,5)P3 and generating PI(3,4)P2 in SF-stimulated BMMCs. We have also shown that SHIP plays a critical role in regulating PKB activity in these cells and that the overall effect of the large increase in PI(3,4,5)P3 (and a decrease in PI(3,4)P2levels) is increased activation of PKB. This is consistent with reports by Liu et al. (
      • Liu Q.
      • Sasaki T.
      • Kozieradzki I.
      • Wakeham A.
      • Itie A.
      • Dumont D.J.
      • Penninger J.M.
      ), Aman et al. (
      • Aman M.J.
      • Lamkin T.D.
      • Okada H.
      • Kurosaki T.
      • Ravichandran K.S.
      ), and Jacobet al. (
      • Jacob A.
      • Cooney D.
      • Tridandapani S.
      • Kelley T.
      • Coggeshall K.M.
      ). However, our data reveal that, while the loss of SHIP enhances PKB activity, the generation of PI(3,4)P2in SHIP−/− BMMCs, although substantially lower than in SHIP+/+ BMMCs in response to SF, is essential to fully activate PKB by enhancing its phosphorylation at serine 473. Our results also suggest that SF-stimulated SHIP−/− and +/+ BMMCs, with their dramatically different PI(3,4,5)P3/PI(3,4)P2 ratios, could prove very useful for future studies comparing the potential role of these two phosphoinositides both in recruitment of target proteins and in regulation of signaling cascades.

      Acknowledgments

      We thank Vivian Lam for excellent technical support and Christine Kelly for help in typing the manuscript. We thank S. Ozaki (University of Utah) for providing the shuttles, Echelon Research Laboratories (Salt Lake City, UT) for providing cargo phosphoinositides, and J. Shope and D. DeWald (Utah State University) for sharing phosphoinositide shuttling protocols prior to publication. We thank Dr. Bayard Clarkson, Memorial Sloan-Kettering Cancer Center, New York, NY for his generous gift of anti-SHIP2 antibody.

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