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A Novel 55-kDa Regulatory Subunit for Phosphatidylinositol 3-Kinase Structurally Similar to p55PIK Is Generated by Alternative Splicing of the p85α Gene (∗)

Open AccessPublished:March 08, 1996DOI:https://doi.org/10.1074/jbc.271.10.5317
      Phosphatidylinositol 3-kinase, which is composed of a 110-kDa catalytic subunit and a regulatory subunit, plays important roles in various cellular signaling mechanisms. We screened a rat brain cDNA expression library with 32P-labeled human IRS-1 protein and cloned cDNAs that were very likely to be generated by alternative splicing of p85α gene products. These cDNAs were demonstrated to encode a 55-kDa protein (p55α) containing two SH2 domains and an inter-SH2 domain of p85α but neither a bcr domain nor a SH3 homology domain. Interestingly, p55α contains a unique 34-amino acid sequence at its NH2 terminus, which is not included in the p85α amino acid sequence. This 34-amino acid portion was revealed to be comparable with p55PIK (p55γ) in length, with a high homology between the two, suggesting that these NH2-terminal domains of p55α and p55γ may have a specific role that p85 does not. The expression of p55α mRNA is most abundant in the brain, but expression is ubiquitous in most rat tissues. Furthermore, it should be noted that the expression of p85α mRNA in muscle is almost undetectably low by Northern blotting with a cDNA probe coding for the p85α SH3 domain, while the expression of p55α can be readily detected. These results suggest that p55α may play an unique regulatory role for phosphatidylinositol 3-kinase in brain and muscle.

      INTRODUCTION

      Phosphatidylinositol 3-kinase (PI 3-kinase) (
      The abbreviations used are: PI 3-kinase
      phosphatidylinositol 3-kinase
      PCR
      polymerase chain reaction
      bp
      base pair(s)
      kb
      kilobase(s)
      SH2
      Src homology 2
      nSH2
      N-terminal Src homology 2
      cSH2
      C-terminal Src homology 2
      SH3
      Src homology 3
      PAGE
      polyacrylamide gel electrophoresis
      HA
      hemagglutinin
      bcr
      breakpoint cluster region.
      ) (
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Kapellar R.
      • Cantley L.C.
      ) has been implicated in the regulation of various cellular activities, including proliferation (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Gold M.R.
      • Duronio V.
      • Saxena S.P.
      • Schrader J.W.
      • Aebersold R.
      ), differentiation(
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ), membrane ruffling(
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ), and prevention of apoptosis(
      • Yao R.
      • Cooper G.M.
      ). In addition, PI 3-kinase activation is required for insulin-stimulated glucose transport and insulin-dependent p70S6K activation(
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ). PI 3-kinase is a heterodimeric enzyme consisting of a regulatory subunit (
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Kapellar R.
      • Cantley L.C.
      ,
      • Shibasaki F.
      • Fukui Y.
      • Takenawa T.
      ,
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Shlessinger J.
      ) and a 110-kDa catalytic subunit (p110α, β). Recently, a novel 110-kDa catalytic subunit (p110γ), which is stimulated via Gα and Gβγ, was cloned(
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Dhand R.
      • Nurnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.D.
      • Wetzker R.
      ). For the former type of PI 3-kinase, three regulatory subunit isoforms for PI 3-kinase have been identified. Among them, p55PIK is a unique protein since the SH3 domain and bcr homology domains found in p85α are replaced in p55PIK by a unique 34-residue NH2 terminus (
      • Pons S.
      • Asano T.
      • Glasheen E.
      • Miralpeix M.
      • Zhang Y.
      • Fisher T.L.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.
      ).
      In this study, we isolated a novel alternatively spliced cDNA from the p85α gene by expression screening from a rat brain cDNA library using a 32P-labeled human IRS-1 protein. This cDNA was demonstrated to encode a 55-kDa protein, which was designated p55α, because it is partly identical to p85α. In addition, we suggest changing the name of p55PIK to p55γ to avoid confusion between p55PIK (p55γ) and p55α. Herein, we compare the amino acid sequences of four isoforms of the regulatory subunit of rat PI 3-kinase and show their tissue distributions. These isoforms may be activated by different stimuli and/or at different intercellular locations.

      MATERIALS AND METHODS

      Preparation of Recombinant Human IRS-1

      Human IRS-1 cDNA was obtained by screening the human genomic library using a 32P-labeled DNA fragment. According to the sequence of human IRS-1 reported by Araki et al.(
      • Araki E.
      • Sun X.J.
      • Haag B.I.
      • Chuang L.M.
      • Zhang Y.
      • Yang-Feng T.L.
      • White M.F.
      • Kahn C.R.
      ), oligonucleotides were synthesized as follows: TCAATGCTGCAACAGCAGATGA as a forward primer and TCAGTGCCAGTCTCTTCCTCTCTG as a reverse primer. PCR amplification was performed using these primers, and a 321-bp fragment was obtained from human genomic DNA. A human genomic library (a generous gift from Dr. H. Hirai, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo) was screened using a 32P-labeled 321-bp PCR fragment, and one positive clone was isolated. The coding region of human IRS-1 genomic DNA was subcloned into pBacPAK9 transfer vector, a baculovirus vector (Invitrogen), and the baculovirus was produced according to the manufacturer's instructions. The purification of IRS-1 from Sf-9 cells infected with the baculovirus containing IRS-1 DNA was performed as described previously(
      • Pons S.
      • Asano T.
      • Glasheen E.
      • Miralpeix M.
      • Zhang Y.
      • Fisher T.L.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.
      ). The insulin receptor was partially purified from human placenta on wheat germ agglutinin agarose as described previously(
      • Kasuga M.
      • White M.F.
      • Kahn C.R.
      ). The 32P-labeled IRS-1 probe was prepared by the incubation of IRS-1 with activated insulin receptor in the presence of Mn2+ and 32P-labeled γ-ATP(
      • Pons S.
      • Asano T.
      • Glasheen E.
      • Miralpeix M.
      • Zhang Y.
      • Fisher T.L.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.
      ).

      Expression Screening with Human [32P]IRS-1 Protein

      An oligo(dT)-primed rat brain cDNA library was prepared in UNI-ZAP XR (Stratagene) according to the manufacturer's instructions. Sixty 15-cm diameter plates representing 3,000,000 independent plaques were plated and incubated for 7 h at 37°C. Then, the plates were overlaid with nitrocellulose filters that had been impregnated with 10 mM isopropyl-β-D-thiogalactopyranoside and incubated for 8 h at 37°C. Hybridization of the filters with the [32P]IRS-1 probe and washing were performed as described previously(
      • Pons S.
      • Asano T.
      • Glasheen E.
      • Miralpeix M.
      • Zhang Y.
      • Fisher T.L.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.
      ). The cDNA inserts in pBluescript were prepared by in vivo excision according to the manufacturer's instructions (Stratagene). The nucleotide sequences were determined using an ABI automatic sequencer.

      Northern Blotting

      Northern blotting was performed using a commercially available filter made by Clontech (Palo Alto, CA). The 1-663-nucleotide sequence of p85α, 1011-2175 of p85α, 1-2170 of p85β, 96-1381 of p55γ, and 1-159 of p55α were labeled with [32P]dCTP and used as probes. The filter was hybridized and washed according to the manufacturer's instructions (Clontech). Autoradiography was performed at −80°C for 12-48 h, and the radioactivities of the bands obtained were measured using a BAS2000 (Fuji).

      Preparation of the Antibodies

      An antibody against the whole p85α molecule (αp85PAN-UBI) was purchased from UBI. An anti-p85α specific antibody (αp85αSH3) was prepared by immunizing rabbits with a 36-amino acid synthetic peptide based on the SH3 domain of p85α (HLGDILTVNKGSLVALGFSDGQEARPEDIGWLNGYN, amino acid residues 25-60). An anti-p55α antibody (αp55α) was raised against a 26-amino acid synthetic peptide in the unique NH2-terminal region of p55α (YTTVWTMEDLDLECAKTDINCGTDLM, amino acid residues 2-27). An anti-p55γ antibody (αp55γ) was raised against an 18-amino acid synthetic peptide in its NH2-terminal portion (DDADWREVMMPYSTELIF amino acid residues 11-28). These peptides were coupled to keyhole limpet hemocyanin, and rabbits were then inoculated with the peptides. The antisera were affinity-purified with Affi-Gel 10 covalently coupled with the corresponding peptides(
      • Oka Y.
      • Asano T.
      • Shibisaki Y.
      • Kasuga M.
      • Kanazawa Y.
      • Takaku F.
      ).
      To confirm the specificity of these antibodies, p85α, p85β, p55γ, and p55α were expressed in Sf-9 cells using the baculovirus system. These cDNAs coding the full amino acid sequence and the HA tag amino acid sequence (YPYDVPDYA) at each C terminus were subcloned into pBacPAK9 transfer vector, and the baculoviruses were prepared according to the manufacturer's instructions (Clontech). The Sf-9 cells infected with baculoviruses containing each of the four isoforms were cultured for 48 h and lysed in Laemmli buffer. The samples were subjected to SDS-PAGE, and immunoblotting was performed as described previously (
      • Oka Y.
      • Asano T.
      • Shibisaki Y.
      • Kasuga M.
      • Kanazawa Y.
      • Takaku F.
      ).

      Immunoblotting of p85α, p55α, and p55γ Expressed in Rat Brain

      Rat brain was homogenized in ice-cold lysis buffer (1/10, w/v) containing 50 mM Hepes (pH 7.5), 137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 2 mM EDTA, 1% Nonidet P-40, 10% glycerol, 2 mg/ml aprotinin, and 34 mg/ml phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14,000 × g for 60 min and incubated for 2 h at 4°C with αp85PAN-UBI covalently coupled with protein A-Sepharose beads, which were also purchased from Upstate Biotechnology Inc. The beads were washed three times in lysis buffer, boiled in Laemmli buffer, and then removed by centrifugation. The supernatants were subjected to SDS-PAGE, and immunoblotting was performed as described previously(
      • Oka Y.
      • Asano T.
      • Shibisaki Y.
      • Kasuga M.
      • Kanazawa Y.
      • Takaku F.
      ).

      PI 3-Kinase Assay

      Rat brain was homogenized in ice-cold lysis buffer containing 20 mM Tris (pH 7.5), 137 mM NaCl, 1 mM CaCl2, 10 mg of aprotinin/ml, and 1 mM phenylmethylsulfonyl fluoride. Lysates were extracted by centrifugation at 14,000 × g for 10 min and incubated with control antibody, αp85PAN-UBI, αp85αSH3, or αp55α for 3 h at 4°C. Protein A-Sepharose beads (Sigma) were used to precipitate the immune complexes. The level of PI 3-kinase activity in immunocomplexes was determined as described previously(
      • Pons S.
      • Asano T.
      • Glasheen E.
      • Miralpeix M.
      • Zhang Y.
      • Fisher T.L.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.
      ).

      RESULTS AND DISCUSSION

      A human IRS-1 gene was successfully cloned from a human genomic library, and the complete nucleotide sequence of its coding region was determined. In comparison with the sequence reported by Araki et al.(
      • Araki E.
      • Sun X.J.
      • Haag B.I.
      • Chuang L.M.
      • Zhang Y.
      • Yang-Feng T.L.
      • White M.F.
      • Kahn C.R.
      ), two nucleotides were revealed to be different in our IRS-1 nucleotide sequence (C to G at 2166 bp and A to G at 3432 bp). The C to G change at 2166 bp caused a change in the amino acid sequence (C to W at 382). As these differences were thought to be due to polymorphism, we prepared recombinant IRS-1 protein using a baculovirus containing this IRS-1 DNA.
      A rat brain cDNA expression library was screened with 32P-labeled recombinant IRS-1, and 81 positive independent clones were isolated after three or four rounds of screening. They included cDNAs containing complete coding regions of p85α, p85β, and p55γ, of which nucleotide sequences were determined. In addition, we obtained three independent cDNAs containing the nucleotide sequence coding for the NH2-terminal SH2 domain of p85α and previously undocumented 166-nucleotide sequence at its 5′-upstream side. These cDNAs contained an open reading frame of 1362 nucleotides, and the deduced amino acid sequence is shown in Fig. 1. The presence of this mRNA in rat brain was confirmed by reverse transcription PCR using the 5′-primer in the newly identified nucleotide sequence and the 3′-primer in the nSH2 domain or in the cSH2 domain found in p85α cDNA (data not shown). We designated this putative protein p55α on the basis of its molecular weight. p55α contains two SH2 domains and an inter-SH2 domain, which are identical to those of p85α. Thus, p55α mRNA appears to be transcribed by alternative splicing from the p85α gene. The SH3 domain and bcr homology domain found in p85α are replaced in p55α by a unique 34-residue NH2 terminus followed by a conserved proline-rich motif (PPALPPKPPKP). Interestingly, this 34-residue region of p55α is comparable in length to the corresponding NH2-terminal portion of p55γ(
      • Pons S.
      • Asano T.
      • Glasheen E.
      • Miralpeix M.
      • Zhang Y.
      • Fisher T.L.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.
      ), and 16 of the 34 amino acids are identical in the two peptides. These conserved sequences suggest that their unique NH2-terminal portion may have a specific functional role, which p85 does not. Further study is needed to resolve this issue.
      Figure thumbnail gr1
      Figure 1:Alignment of amino acid sequences of p85α, p55α, p55γ, and p85β. The cDNAs coding for the complete peptides of p85α, p55α, p55γ, and p85β were isolated by screening a rat brain expression cDNA library with 32P-labeled IRS-1 protein probe. The nucleotide sequences were determined with an ABI automatic sequencer. The amino acid residues for each protein, with the addition of gaps(-) to optimize the alignment, are numbered to the right of each sequence. Two SH2, the bcr homology, the proline-rich, and the SH3 domains are boxed.
      The levels of expression of p85α, p55α, p55γ, and p85β mRNAs in various rat tissues were investigated, and the results are shown in Fig. 2. Northern blotting with a 5′-unique 159-nucleotide sequence located in the 5′-untranslated region and a coding region for the NH2-terminal 25-amino acid sequence in the NH2 terminus of p55α, neither of which is included in the p85α cDNA nucleotide sequence, revealed three mRNA species of 6.0, 4.2, and 2.8 kb in the brain (Fig. 2B). Among them, the 4.2-kb band was also detected in all of other tissues examined. Northern blotting with nucleotides coding for the p85α SH3 domain revealed two mRNA species of 7.7 and 4.2 kb (Fig. 2A). In addition, the cDNA probe coding for the p85α/p55α nSH2 domains was also used for Northern blotting, and four mRNA species of 7.7, 6.0, 4.2, and 2.8 kb were observed (Fig. 2C). The 4.2-kb band was detected on all Northern blots, and the intensities of this band were compared in various tissues. The amount of the 4.2-kb mRNA detected with Northern blotting using a cDNA probe coding for the p85α/p55α nSH2 domain is thought to be the sum of the amounts of the p55α and p85α mRNAs. The intensity of the 4.2-kb band among various tissues observed in blotting utilizing a cDNA probe coding for the p85α/p55α nSH2 domain was revealed to be similar to that obtained with a cDNA probe coding for the p85α SH3 domain and differed significantly from that obtained with the p55α 5′-unique cDNA probe. This result may suggest that p85α mRNA is expressed more abundantly than p55α mRNA in most tissues, with the apparent exceptions of brain and skeletal muscle. However, it should be noted that in skeletal muscle the p85α mRNA expression level is almost undetectably low, while p55α mRNA can be readily detected. In muscle, the activation of PI 3-kinase is presumed to be involved in insulin-stimulated glucose uptake through the translocation of GLUT4 to the plasma membrane(
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ). Therefore, it might be possible that p55α plays a more important role than p85α in the stimulation of glucose uptake by skeletal muscle.
      Figure thumbnail gr2
      Figure 2:Northern blotting of p85α, p55α, p55γ, and p85β mRNAs in various rat tissues. Rat multiple tissue Northern blot was obtained from Clontech and used for the detection of mRNA. 32P-Labeled cDNA probes encoding nucleotides 1-663 of p85α (panel A), 1-159 of p55α (panel B), 1011-2175 of p85α (same as nucleotides 201-1365 of p55α) (panel C), 96-1381 of p55γ (panel D), and nucleotides 1-2170 of p85β (panel E) were hybridized and washed according to the manufacturer's instruction (Clontech). The intensity of the 4.2-kb bands was measured by using a BAS2000 (Fuji).
      In brain, both p55α and p55γ mRNAs are expressed abundantly, as are those of p85α, suggesting that these regulatory subunits having neither bcr homology nor SH3 domains may have a function(s) different from that of p85. PI 3-kinase appears to be important, first, in that its activation is essential for neurite elongation of rat PC-12 cells, and in addition, VPS34, a yeast PI 3-kinase homologue, was shown to be involved in vacuolar protein sorting(
      • Schu P.V.
      • Kaoru T.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emr S.D.
      ). Thus, PI 3-kinase may play an essential role in the secretion of neurotransmitters via regulation of vesicle sorting in the brain. Taken together, one or more of these four regulatory subunits might be essential for neuronal differentiation, while the others may be involved in the secretion of neurotransmitters.
      Unlike p85α and p55α, p55γ and p85β genes generate only one mRNA species each, of 5.8 and 3.4 kb, respectively (Fig. 2, D and E), suggesting that no other mRNAs are generated by alternative splicing of p55γ and p85β gene products.
      In order to detect the endogeneous p85α, p55α, and p55γ proteins in rat tissues, we prepared specific antibodies against each of the three. These antibodies did not recognize different isoforms of regulatory subunits, produced in the Sf-9 cell experiment using the baculovirus expression system (Fig. 3). As shown in Fig. 3A, by immunoblotting using the anti-HA antibody (12CA5), the electrophoretic mobility of p55α is essentially the same as that of p55γ. The rat brain lysates immunoabsorbed by the beads covalently coupled with αp85PAN-UBI, which recognize all p85α, p55α, p55γ, and p85β expressed in Sf-9 cells because of the highly conserved amino acid sequence of these peptides (data not shown), were subjected to SDS-PAGE and immunoblotted with control antibody, αp85PAN-UBI, αp85αSH3, αp55α, and αp55γ (Fig. 3E). The immunoblot obtained with αp85PAN-UBI revealed the two bands of 85 and 55 kDa, while that obtained with αp85αSH3 showed only the 85-kDa band. In contrast, αp55α and αp55γ both showed the 55-kDa band. These results indicate the expression of these isoforms in brain.
      Figure thumbnail gr3
      Figure 3:A-D, immunoblotting of p85α, p85β, p55γ, and p55α expressed in Sf-9 cells. To confirm the specificities of the antibodies, p85α, p85β, p55γ, and p55α were expressed in Sf-9 cells using a baculovirus system. The cDNAs coding for the full amino acid sequences and the HA-tag amino acid sequence at the C termini were subcloned into pBacPAK9 transfer vector (Clontech), and the baculoviruses were prepared according to the manufacturer's instructions. The Sf-9 cells infected with baculoviruses containing one of each of the four isoforms were cultured for 48 h and lysed in Laemmli buffer. The samples were subjected to SDS-PAGE, and immunoblotting was performed using anti-HA antibody (panel A), αp85αSH3 (panel B), αp55α (panel C), or αp55γ (panel D), as described previously(
      • Oka Y.
      • Asano T.
      • Shibisaki Y.
      • Kasuga M.
      • Kanazawa Y.
      • Takaku F.
      ). Lane 1, control Sf-9 cells; lanes 2-5, Sf-9 cells expressing p55α, p55γ, p85α, and p85β, respectively. E, immunoblotting of p85α, p55α, and p55γ. Rat brain was homogenized and solubilized in the lysis buffer. Supernatants were collected after centrifugation and incubated with beads coupled with antibody against the whole p85α molecule (Upstate Biotechnology Inc.). The beads were washed three times and resuspended in Laemmli buffer. The eluants from the beads were electrophoresed and immunoblotted with control antibody, αp85PAN-UBI, αp85αSH3, αp55α, or αp55γ. F, PI 3-kinase activities in immunoprecipitates obtained with control antibody, αp85PAN-UBI, αp85αSH3, or αp55α. Rat brain was solubilized and the supernatants, obtained by centrifugation, were incubated with control antibody (lane 1), αp85PAN-UBI (lane 2), αp85αSH3 (lane 3), or αp55α (lane 4). The PI 3-kinase activities of these immunoprecipitates were measured as described under “Materials and Methods.”
      Finally, to determine whether or not p55α is associated with PI 3-kinase activity, as in the case of p85α, we immunoprecipitated the rat brain soluble fraction with each control antibody, αp85PAN-UBI, αp85αSH3, or αp55α. PI 3-kinase activities in these immunoprecipitates were measured (Fig. 3F). The immunoprecipitates obtained with αp85PAN-UBI, αp85αSH3, or αp55α were demonstrated to contain significant PI 3-kinase activity, as compared with the control antibody immunoprecipitate. This result strongly suggests that p55α also exists as a heterodimer with a p110 catalytic subunit and that it functions as a regulatory subunit of PI 3-kinase.
      In this study, we showed that there are at least four isoforms of the regulatory subunit for PI 3-kinase. All of the four isoforms contain two SH2 domains and the binding site for association with the p110 catalytic subunit, suggesting that these regulatory subunits of PI 3-kinase interact with phosphotyrosine residues on the receptors or receptor substrates through one or both of their SH2 domains, resulting in activation of the p110 catalytic subunit. However, SH3 and bcr homology domains found in p85α or -β are replaced in p55α or -γ by unique 34-residue NH2 termini. Although the functional roles of SH3 domain have not been understood yet, the association between SH3 domain and proline-rich segments in various signaling proteins (i.e. dynamin(
      • Gout I.
      • Dhand R.
      • Hiles I.D.
      • Fry M.J.
      • Panayotou G.
      • Das P.
      • Truong O.
      • Totty N.F.
      • Hsuan J.
      • Booker G.W.
      • Campbell I.D.
      • Waterfield M.D.
      ), paxillin(
      • Weng Z.
      • Taylor J.A.
      • Turner C.E.
      • Brugge J.S.
      • Seidel-Dugan C.
      ), hSOS1 (
      • Chardin P.
      • Camonis J.H.
      • Gale N.W.
      • Van Aelst L.
      • Schlessinger J.
      • Wigler M.H.
      • Bar-Sagi D.
      ), p85 subunit of PI 3-kinase(
      • Prasad K.V.S.
      • Janssen O.
      • Kapellar R.
      • Raab M.
      • Cantly L.C.
      • Rudd C.E.
      )) is reported. Thus, the differences in the NH2-terminal region observed among the regulatory subunit isoforms may contribute to differences in subcellular distributions and/or to varying degrees of PI 3-kinase activation in response to various growth factor receptors and oncogenic products.
      In summary, we have identified a novel alternatively spliced regulatory subunit, which may have important functions in brain and muscle. Our future studies will focus on the variety of possible functions mediated by differences in the NH2terminal portions of the regulatory subunits of PI 3-kinase.

      Acknowledgments

      We thank Dr. Hisamaru Hirai, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, for the generous gift of a human genomic library.

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