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p110δ, a Novel Phosphatidylinositol 3-Kinase Catalytic Subunit That Associates with p85 and Is Expressed Predominantly in Leukocytes*

Open AccessPublished:August 01, 1997DOI:https://doi.org/10.1074/jbc.272.31.19236
      We have identified a novel p110 isoform of phosphatidylinositol 3-kinase from human leukocytes that we have termed p110δ. In addition, we have independently isolated p110δ from a mouse embryo library on the basis of its ability to interact with Ha-RasV12 in the yeast two-hybrid system. This unique isoform contains all of the conserved structural features characteristic of the p110 family. Recombinant p110δ phosphorylates phosphatidylinositol and coimmunoprecipitates with p85. However, in contrast to previously described p110 subunits, p110δ is expressed in a tissue-restricted fashion; it is expressed at high levels in lymphocytes and lymphoid tissues and may therefore play a role in phosphatidylinositol 3-kinase-mediated signaling in the immune system.
      Phosphatidylinositol (PI)
      The abbreviations used are: PI, phosphatidylinositol; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; PBMC, peripheral blood mononuclear cells; RT, reverse transcription; PIPES, 1,4-piperazinediethanesulfonic acid; Rip, Ras-interacting protein.
      1The abbreviations used are: PI, phosphatidylinositol; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; PBMC, peripheral blood mononuclear cells; RT, reverse transcription; PIPES, 1,4-piperazinediethanesulfonic acid; Rip, Ras-interacting protein.
      3-kinase was originally identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases that phosphorylates PI and its phosphorylated derivatives at the 3′-hydroxyl of the inositol ring (
      • Panayatou G.
      • Waterfield M.D.
      ). The purification and subsequent molecular cloning of PI 3-kinase revealed that it is a heterodimer consisting of p85 and p110 subunits (
      • Otsu M.
      • Hiles I.D.
      • Gout I.
      • Fry M.J.
      • Ruiz-Larrea F.
      • Panayatou G.
      • Thompson A.
      • Dhand R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Courtnidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayatou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtnidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ,
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ).
      The p85 subunit acts to localize PI 3-kinase activity to the plasma membrane by virtue of the interaction of its SH2 domain with phosphorylated tyrosine residues (present in an appropriate local sequence context) in target proteins (
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ). Two isoforms of p85 have been identified: p85α, which is ubiquitously expressed, and p85β, which is primarily found in brain and lymphoid tissues (
      • Volinia S.
      • Patracchini P.
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Calzari E.
      • Bernardi F.
      • Rooke L.
      • Waterfield M.D.
      ). The p110 subunit contains the catalytic domain of PI 3-kinase, and three isoforms of p110 have thus far been reported (α, β, and γ) (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayatou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtnidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ,
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanov S.
      • Vanhaeseroeck B.
      • Dhand R.
      • Nurnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.W.
      • Wetzker R.
      ). The identification of p110γ revealed additional complexity within this family of enzymes. p110γ is most closely related to p110α and β (45–48% identity in the catalytic domain) but does not make use of p85 as a targeting subunit. p110γ contains an additional domain termed a pleckstrin homology domain near the amino terminus. The pleckstrin homology domain allows interaction with the βγ subunits of heterotrimeric G proteins that appears to regulate its activity and subcellular localization (
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanov S.
      • Vanhaeseroeck B.
      • Dhand R.
      • Nurnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.W.
      • Wetzker R.
      ).
      Additional members of this growing gene family include more distantly related lipid and protein kinases such as Vps34, TOR1, and TOR2 ofSaccharomyces cerevisiae (and their mammalian homologues such as FRAP and mTOR), the human ataxia telangiectasia gene product, and the catalytic subunit of DNA-dependent protein kinase ().
      The levels of phosphatidylinositol-3,4,5-triphosphate, the primary product of PI 3-kinase activation, are elevated upon treatment of cells with a wide variety of agonists (
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ). This observation has implicated PI 3-kinase activation in a diverse range of cellular responses including cell growth, differentiation, and apoptosis (
      • Panayatou G.
      • Waterfield M.D.
      ,
      • Parker P.J.
      ,
      • Yao R.
      • Cooper G.M.
      ). The downstream targets of the phosphorylated lipids generated following PI 3-kinase activation have not been well characterized. However some isoforms of protein kinase C are directly activated by phosphatidylinositol-3,4,5-triphosphate in vitro. The protein kinase C-related protein kinase AKT has also been shown to be activated by PI 3-kinase, although the mechanism of this has yet to be determined (
      • Downward J.
      ).
      PI 3-kinase also appears to be involved in a number of aspects of leukocyte activation. PI 3-kinase physically associates with the cytoplasmic domain of CD28, which is an important co-stimulatory molecule for the activation of T cells in response to antigen (
      • Pages F.
      • Rageneau M.
      • Rottapel R.
      • Truneh A.
      • Nunes J.
      • Imbert J.
      • Olive D.
      , ). Activation of T cells through CD28 lowers the threshold for activation by antigen and increases the magnitude and duration of the proliferative response. These effects are linked to increases in the transcription of a number of genes including the T cell growth factor interleukin 2 (
      • Fraser J.D.
      • Irving B.A.
      • Creabtree G.R.
      • Weiss A.
      ). Mutation of CD28 such that it can no longer interact with PI 3-kinase leads to a failure to initiate interleukin-2 production, suggesting a critical role for PI 3-kinase in T cell activation (
      • Pages F.
      • Rageneau M.
      • Rottapel R.
      • Truneh A.
      • Nunes J.
      • Imbert J.
      • Olive D.
      ). Based on studies using the PI 3-kinase inhibitor wortmannin, there is evidence that PI 3-kinase(s) is also required for some aspects of leukocyte signaling through G protein-coupled receptors (
      • Thelen M.
      • Wymann M.P.
      • Langen H.
      ).
      We report here the cloning of novel human and murine p110 isoforms (p110δ) with a highly restricted pattern of expression. p110δ is expressed predominantly in leukocytes and may therefore play a role in PI 3-kinase-mediated signaling in the immune system.

      RESULTS AND DISCUSSION

      Using a strategy based on amplification of conserved PI 3-kinase sequences, we have identified a novel human member of this family that we have termed p110δ. A combination of cDNA library screening and 5′ RACE PCR has led to the identification of cDNAs encompassing the complete coding region of p110δ. The deduced amino acid sequence of p110δ is shown in Fig. 1.
      Figure thumbnail gr1
      Figure 1Deduced amino sequence of human (Hu) and murine (Mu) p110δ. Identical residues are shown by dots, the Ras regulatory domain isunderlined, and the start of the carboxyl-terminal catalytic domain is shown by a ↓.
      In an independent search for mouse Ha-RasV12-interacting proteins (Rips) using the yeast two-hybrid system, we identified two clones related in sequence to p110β: Rip31 and Rip36. Using a PCR-based strategy and gene-specific oligonucleotide primers derived from the Rip36 sequence, a full-length cDNA was isolated (see “Materials and Methods”). Sequence analysis suggests that this clone is the murine p110δ, since it shares 94% amino acid sequence identity with human p110δ (compared with 56% identity between human p110δ and β; an alignment of human and mouse p110δ is shown in Fig. 1) and has a similar pattern of expression in vivo (see below).
      The sequences of human and murine p110δ include open reading frames predicted to encode for proteins of 1044 and 1043 amino acids, respectively, with an expected molecular mass of 119,505 Da for the human clone (∼120 kDa). The sequences around the predicted initiating methionines are in good agreement with that required for optimal translational initiation (
      • Kozak M.
      ). The presence of stop codons in the 5′-untranslated sequence is consistent with isolation of the complete coding region of p110δ (data not shown). Consistent with the predicted size of the encoded translation product, Western blotting of immunoprecipitates from COS cells transiently transfected with an epitope-tagged form of human p110δ detected a protein of ∼110 kDa (see below).
      Comparison of the sequence of the carboxyl-terminal catalytic domain of p110δ with those of other PI 3-kinases reveals that it is most closely related to p110β. p110δ is 72% identical to p110β in this region and is less closely related to p110α (49%) or p110γ (45%), whereas cpk/p170 and the yeast Vps34 protein show the lowest homology (31 and 32%, respectively). This is confirmed by dendrogram analysis; p110β and p110δ form a distinct sub-branch of the PI 3-kinase family (Fig. 2). The distantly related ataxia telangiectasia gene product and the catalytic subunit of DNA-dependent protein kinase have been included for comparison (Fig. 2). These proteins are structurally related to PI 3-kinases and have protein kinase activity (
      • Keegan K.S.
      • Holtzman D.H.
      • Plug A.W.
      • Christienson E.R.
      • Brainerd E.E.
      • Flaggs G.
      • Bentley N.J.
      • Taylor E.M.
      • Meyn M.S.
      • Moss S.B.
      • Carr A.M.
      • Ashley T.
      • Hoekstra M.F.
      ) but have not yet been shown to possess lipid kinase activity (
      • Hartley K.O.
      • Gell D.
      • Smith G.C.M.
      • Zhang H.
      • Divecha N.
      • Connelly M.A.
      • Admon A.
      • Lees-Miller S.P.
      • Anderson C.W.
      • Jackson S.P.
      ,
      • Savitsky K.
      • Bar-Shira A.
      • Gilad S.
      • Rotman G.
      • Ziv Y.
      • Vanagaite L.
      • Tagle D.L.
      • Smith S.
      • Uziel T.
      • Sfez S.
      • Ashkenazi M.
      • Pecker I.
      • Frydman M.
      • Harnik R.
      • Patenjali S.R.
      • Simmons A.
      • Clines G.A.
      • Sartiel A.
      • Gatti R.A.
      • Chessa L.
      • Sanal O.
      • Lavin M.F.
      • Jaspers N.G.J.
      • Taylor M.R.
      • Arlett C.F.
      • Miki T.
      • Weissman S.M.
      • Lovett M.
      • Collins F.S.
      • Shiloh Y.
      ).
      Figure thumbnail gr2
      Figure 2Dendrogram analysis of the PI 3-kinase family. The analysis was performed based on the predicted catalytic domains corresponding to the C-terminal 320 amino acids.DNA PK CS CS, catalytic subunit of DNA-dependent protein kinase; ATM, ataxia telangiectasia gene product.
      p110α and p110β form heterodimers with a common p85 subunit (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayatou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtnidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ,
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ,
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ). We examined the association of recombinant p110δ with p85. When either epitope-tagged human (Fig. 3, panel B) or mouse (Fig. 4, lane 4) p110δ was expressed in COS or 293T cells and recovered by immunoprecipiation, a 85-kDa protein was detected with p85-specific antiserum in the immunoprecipitates. Thus, both human and murine p110δ associate with endogenous p85 after transfection into COS or 293T cells, respectively. Association of human p110δ with p85 could also be detected following expression of epitope-tagged p110δ in the lymphoid cell line Jurkat (data not shown). The association of p110α, p110β, and p110δ with p85 is consistent with the presence of a conserved p85 interaction domain at the amino terminus of these isoforms. This region is lacking in p110γ, which is targeted to the plasma membrane via its interaction with the β/γ subunits of heterotrimeric G proteins. This interaction is dependent on a p110γ-specific adaptor protein, p101 (
      • Stephens L.R.
      • Eguinoa A.
      • Erdjument-Bromage H.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.S.
      • Thelen M.
      • Cadwallader K.
      • Tempst P.
      • Hawkins P.T.
      ).
      Figure thumbnail gr3
      Figure 3Human p110δ associates with p85α.FLAG-tagged p110δ was expressed in COS cells and immunoprecipitated with the anti-FLAG M2 antibody. Immunoprecipitates were analyzed by 8% SDS-polyacrylamide gel electrophoresis followed by immunoblotting using either anti-FLAG M2 or anti-p85α. Panel A shows coimmunoprecipitation of p110δ with p85α. The control lane is a lysate from the Jurkat cell line that constitutively expresses p85α. Panel B shows that anti-FLAG M2 recognizes an ∼110-kDa protein in immunoprecipitates from p110δ-transfected cells.
      Figure thumbnail gr4
      Figure 4Murine p110δ associates with p85.Glutathione S-transferase-tagged murine p110δ was expressed in 293T cells, and the glutathione S-transferase fusion protein was recovered by incubation with glutathione-Sepharose resin. Proteins bound to the resin were analyzed by 12.5% SDS-polyacrylamide gel electrophoresis followed by immunoblotting using an anti-glutathione S-transferase antibody or anti-p85.Lanes 1 and 2 show expression of the fusion protein. Lanes 3 and 4 show that endogenous p85α associates with the glutathione S-transferase-tagged murine p110δ. An arrow marks the position of the p85 protein.
      It has been demonstrated that PI 3-kinase is an important intermediate in the Ras pathway (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ). A specific region at the amino terminus of the p110α subunit, residues 133–314, termed the Ras regulatory domain (underlined in Fig. 1), is responsible for this interaction (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ). Comparison of the sequence of both human and murine p110δ with other p110 subunits indicates that this region is also conserved in p110δ, including a lysine residue (residues 227 of p110α and 223 of p110δ), which has been shown to be essential for physical association with Ras (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ). Moreover, a relatively short amino acid domain of p110δ (amino acids 141–291, the amino acids of p110δ encoded by the Rip36 clone) is sufficient to interact with Ha-RasV12 in vivo in the yeast two-hybrid system and further delineates the Ras regulatory domain to a 151-amino acid region. The interaction of Ha-RasV12 with this domain of p110δ requires active Ras and an intact Ras effector domain (data not shown). Thus, p110δ is likely to mediate some of the effects of Ras, although the p110δ Ras-interacting region is less conserved than the putative p85 binding site or catalytic domain.
      Whereas the activation of PI 3-kinase in a wide range of biological systems has been extensively studied, less is known concerning the cell type-specific expression of particular p110 isoforms. Northern blot analysis of the expression of p110δ in human and murine tissues reveals a single transcript of ∼5.4 kilobases (consistent with the size of the composite cDNAs). In humans, the highest levels of expression are seen in PBMC, spleen, and thymus (Fig.5). After prolonged exposure of the autoradiograph, low levels of p110δ expression could be detected in testes, uterus, colon, and small intestine but not in other tissues examined including prostate, heart, brain, and liver (data not shown.) p110δ is also abundantly expressed in adult mouse spleen as well as in testis (Fig.5 B). The elevated expression of p110δ mRNA in mouse but not human testes is noteworthy and may reflect a true difference in expression between species. Alternatively, a number of genes is expressed specifically at elevated levels in postmeiotic haploid cells in the testis. Abnormally sized transcripts that may be more stable than the transcripts found in diploid cells are commonly found. However, it is not clear whether these transcripts are translated (Ref.
      • Sorrentino V.
      • McKinney M.D.
      • Giorgi M.
      • Geremia R.
      • Fleissner E.
      and references cited therein). In the case of p110δ, the RNA expressed in the testis may or may not be translated. If p110δ protein is expressed, then it is possible that the protein has a specific role in development of the male germ line. The restricted expression of p110δ is in contrast to p110α and p110β, which appear to be widely expressed (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayatou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtnidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ).
      Figure thumbnail gr5
      Figure 5Tissue distribution of p110δ. RNA from various human or murine tissues was examined for the expression of p110δ by Northern blotting. A, human tissues hybridized with the human cDNA probe. B, murine tissues hybridized with the murine cDNA probe. H, heart; B, brain; S, spleen;, Lu, lung; M, muscle; K, kidney; T, testes; L, liver.
      To test whether p110δ has PI 3-kinase activity, immunoprecipitates from COS cells transfected with epitope-tagged p110δ were incubated with [32P]ATP and phosphatidylinositol, and the radiolabeled phospholipids were resolved by chromatography. A product was detected that migrates slightly slower than the PI 4-phosphate (PIP) standard, consistent with the generation of PI 3-phosphate (
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ) (Fig. 6). This enzyme activity was sensitive to the PI 3-kinase inhibitor wortmannin (data not shown). Similar results were obtained when purified phosphatidylinositol was used as a substrate (data not shown). Whereas these results demonstrate that the cDNA clone that we isolated encodes a functional PI 3-kinase, it cannot be assumed that thein vitro substrate specificity of a particular isoform reflects its activity on membrane lipids in intact cells (
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ).
      Figure thumbnail gr6
      Figure 6p110δ has PI 3-kinase activity.Anti-p110δ immunoprecipitates were assayed for PI 3-kinase activity using phosphatidylinositol as a substrate. Reactions were performed as described under “Materials and Methods.” The reaction products were resolved by thin layer chromatography followed by autoradiography. PI 3-kinase activity is detected in immunoprecipitates from p110δ-transfected (but not vector control) cells. The position of the PI 4-phosphate (PIP) standard is shown. ORI, origin.
      The interaction of multiple p110 catalytic subunit isoforms, p110α, p110β, and p110δ, with a common adaptor protein, p85, suggests that the nature of the phosphorylated lipids generated in response to a particular agonist may be regulated at least in part by the cell/tissue-specific expression of the different isoforms of the catalytic subunit. In cells such as leukocytes, where it is likely that multiple p110 isoforms are expressed, it will be of interest to determine the relative contribution made by these multiple isoforms to processes such as cell activation and migration.

      ACKNOWLEDGEMENTS

      We thank David Turner, Bart Vanhaesebroeck, and Pablo Rodriguez-Viciana for discussions and Johnny Stine for help in preparing the figures.

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