©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel 55-kDa Regulatory Subunit for Phosphatidylinositol 3-Kinase Structurally Similar to p55PIK Is Generated by Alternative Splicing of the p85 Gene (*)

(Received for publication, November 7, 1995)

Kouichi Inukai (1) Motonobu Anai (1) Eric Van Breda (1) Toshio Hosaka (1) Hideki Katagiri Makoto Funaki Yasushi Fukushima Takehide Ogihara Yoshio Yazaki Masatoshi Kikuchi (1) Yoshitomo Oka (2) Tomoichiro Asano (§)

From the  (1)Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan, The Institute for Adult Disease, Asahi Life Foundation, 1-9-14, Nishishinjuku, Shinjuku-ku, Tokyo 160, Japan, and the (2)Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1144, Kogushi, Ube, Yamaguchi 755, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 P-labeled human IRS-1 protein and cloned cDNAs that were very likely to be generated by alternative splicing of p85alpha gene products. These cDNAs were demonstrated to encode a 55-kDa protein (p55alpha) containing two SH2 domains and an inter-SH2 domain of p85alpha but neither a bcr domain nor a SH3 homology domain. Interestingly, p55alpha contains a unique 34-amino acid sequence at its NH(2) terminus, which is not included in the p85alpha amino acid sequence. This 34-amino acid portion was revealed to be comparable with p55PIK (p55) in length, with a high homology between the two, suggesting that these NH(2)-terminal domains of p55alpha and p55 may have a specific role that p85 does not. The expression of p55alpha mRNA is most abundant in the brain, but expression is ubiquitous in most rat tissues. Furthermore, it should be noted that the expression of p85alpha mRNA in muscle is almost undetectably low by Northern blotting with a cDNA probe coding for the p85alpha SH3 domain, while the expression of p55alpha can be readily detected. These results suggest that p55alpha may play an unique regulatory role for phosphatidylinositol 3-kinase in brain and muscle.


INTRODUCTION

Phosphatidylinositol 3-kinase (PI 3-kinase) (^1)(1, 2) has been implicated in the regulation of various cellular activities, including proliferation (3, 4) , differentiation(5) , membrane ruffling(6) , and prevention of apoptosis(7) . In addition, PI 3-kinase activation is required for insulin-stimulated glucose transport and insulin-dependent p70S6K activation(3) . PI 3-kinase is a heterodimeric enzyme consisting of a regulatory subunit (1, 2, 8, 9) and a 110-kDa catalytic subunit (p110alpha, beta). Recently, a novel 110-kDa catalytic subunit (p110), which is stimulated via Galpha and Gbeta, was cloned(10) . 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 p85alpha are replaced in p55PIK by a unique 34-residue NH(2) terminus (11) .

In this study, we isolated a novel alternatively spliced cDNA from the p85alpha gene by expression screening from a rat brain cDNA library using a P-labeled human IRS-1 protein. This cDNA was demonstrated to encode a 55-kDa protein, which was designated p55alpha, because it is partly identical to p85alpha. In addition, we suggest changing the name of p55PIK to p55 to avoid confusion between p55PIK (p55) and p55alpha. 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 P-labeled DNA fragment. According to the sequence of human IRS-1 reported by Araki et al.(12) , 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 P-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(11) . The insulin receptor was partially purified from human placenta on wheat germ agglutinin agarose as described previously(13) . The P-labeled IRS-1 probe was prepared by the incubation of IRS-1 with activated insulin receptor in the presence of Mn and P-labeled -ATP(11) .

Expression Screening with Human [P]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-beta-D-thiogalactopyranoside and incubated for 8 h at 37 °C. Hybridization of the filters with the [P]IRS-1 probe and washing were performed as described previously(11) . 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 p85alpha, 1011-2175 of p85alpha, 1-2170 of p85beta, 96-1381 of p55, and 1-159 of p55alpha were labeled with [P]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 p85alpha molecule (alphap85) was purchased from UBI. An anti-p85alpha specific antibody (alphap85alpha) was prepared by immunizing rabbits with a 36-amino acid synthetic peptide based on the SH3 domain of p85alpha (HLGDILTVNKGSLVALGFSDGQEARPEDIGWLNGYN, amino acid residues 25-60). An anti-p55alpha antibody (alphap55alpha) was raised against a 26-amino acid synthetic peptide in the unique NH(2)-terminal region of p55alpha (YTTVWTMEDLDLECAKTDINCGTDLM, amino acid residues 2-27). An anti-p55 antibody (alphap55) was raised against an 18-amino acid synthetic peptide in its NH(2)-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(14) .

To confirm the specificity of these antibodies, p85alpha, p85beta, p55, and p55alpha 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 (14) .

Immunoblotting of p85alpha, p55alpha, and p55 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 CaCl(2), 1 mM MgCl(2), 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 times g for 60 min and incubated for 2 h at 4 °C with alphap85 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(14) .

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 CaCl(2), 10 mg of aprotinin/ml, and 1 mM phenylmethylsulfonyl fluoride. Lysates were extracted by centrifugation at 14,000 times g for 10 min and incubated with control antibody, alphap85, alphap85alpha, or alphap55alpha 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(11) .


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.(12) , 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 P-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 p85alpha, p85beta, and p55, of which nucleotide sequences were determined. In addition, we obtained three independent cDNAs containing the nucleotide sequence coding for the NH(2)-terminal SH2 domain of p85alpha 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 p85alpha cDNA (data not shown). We designated this putative protein p55alpha on the basis of its molecular weight. p55alpha contains two SH2 domains and an inter-SH2 domain, which are identical to those of p85alpha. Thus, p55alpha mRNA appears to be transcribed by alternative splicing from the p85alpha gene. The SH3 domain and bcr homology domain found in p85alpha are replaced in p55alpha by a unique 34-residue NH(2) terminus followed by a conserved proline-rich motif (PPALPPKPPKP). Interestingly, this 34-residue region of p55alpha is comparable in length to the corresponding NH(2)-terminal portion of p55(11) , and 16 of the 34 amino acids are identical in the two peptides. These conserved sequences suggest that their unique NH(2)-terminal portion may have a specific functional role, which p85 does not. Further study is needed to resolve this issue.


Figure 1: Alignment of amino acid sequences of p85alpha, p55alpha, p55, and p85beta. The cDNAs coding for the complete peptides of p85alpha, p55alpha, p55, and p85beta were isolated by screening a rat brain expression cDNA library with P-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 p85alpha, p55alpha, p55, and p85beta 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 NH(2)-terminal 25-amino acid sequence in the NH(2) terminus of p55alpha, neither of which is included in the p85alpha 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 p85alpha SH3 domain revealed two mRNA species of 7.7 and 4.2 kb (Fig. 2A). In addition, the cDNA probe coding for the p85alpha/p55alpha 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 p85alpha/p55alpha nSH2 domain is thought to be the sum of the amounts of the p55alpha and p85alpha mRNAs. The intensity of the 4.2-kb band among various tissues observed in blotting utilizing a cDNA probe coding for the p85alpha/p55alpha nSH2 domain was revealed to be similar to that obtained with a cDNA probe coding for the p85alpha SH3 domain and differed significantly from that obtained with the p55alpha 5`-unique cDNA probe. This result may suggest that p85alpha mRNA is expressed more abundantly than p55alpha mRNA in most tissues, with the apparent exceptions of brain and skeletal muscle. However, it should be noted that in skeletal muscle the p85alpha mRNA expression level is almost undetectably low, while p55alpha 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(3) . Therefore, it might be possible that p55alpha plays a more important role than p85alpha in the stimulation of glucose uptake by skeletal muscle.


Figure 2: Northern blotting of p85alpha, p55alpha, p55, and p85beta mRNAs in various rat tissues. Rat multiple tissue Northern blot was obtained from Clontech and used for the detection of mRNA. P-Labeled cDNA probes encoding nucleotides 1-663 of p85alpha (panel A), 1-159 of p55alpha (panel B), 1011-2175 of p85alpha (same as nucleotides 201-1365 of p55alpha) (panel C), 96-1381 of p55 (panel D), and nucleotides 1-2170 of p85beta (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 p55alpha and p55 mRNAs are expressed abundantly, as are those of p85alpha, 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(15) . 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 p85alpha and p55alpha, p55 and p85beta 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 p55 and p85beta gene products.

In order to detect the endogeneous p85alpha, p55alpha, and p55 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 p55alpha is essentially the same as that of p55. The rat brain lysates immunoabsorbed by the beads covalently coupled with alphap85, which recognize all p85alpha, p55alpha, p55, and p85beta 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, alphap85, alphap85alpha, alphap55alpha, and alphap55 (Fig. 3E). The immunoblot obtained with alphap85 revealed the two bands of 85 and 55 kDa, while that obtained with alphap85alpha showed only the 85-kDa band. In contrast, alphap55alpha and alphap55 both showed the 55-kDa band. These results indicate the expression of these isoforms in brain.


Figure 3: A-D, immunoblotting of p85alpha, p85beta, p55, and p55alpha expressed in Sf-9 cells. To confirm the specificities of the antibodies, p85alpha, p85beta, p55, and p55alpha 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), alphap85alpha (panel B), alphap55alpha (panel C), or alphap55 (panel D), as described previously(14) . Lane 1, control Sf-9 cells; lanes 2-5, Sf-9 cells expressing p55alpha, p55, p85alpha, and p85beta, respectively. E, immunoblotting of p85alpha, p55alpha, and p55. 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 p85alpha 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, alphap85, alphap85alpha, alphap55alpha, or alphap55. F, PI 3-kinase activities in immunoprecipitates obtained with control antibody, alphap85, alphap85alpha, or alphap55alpha. Rat brain was solubilized and the supernatants, obtained by centrifugation, were incubated with control antibody (lane 1), alphap85 (lane 2), alphap85alpha (lane 3), or alphap55alpha (lane 4). The PI 3-kinase activities of these immunoprecipitates were measured as described under ``Materials and Methods.''



Finally, to determine whether or not p55alpha is associated with PI 3-kinase activity, as in the case of p85alpha, we immunoprecipitated the rat brain soluble fraction with each control antibody, alphap85, alphap85alpha, or alphap55alpha. PI 3-kinase activities in these immunoprecipitates were measured (Fig. 3F). The immunoprecipitates obtained with alphap85, alphap85alpha, or alphap55alpha were demonstrated to contain significant PI 3-kinase activity, as compared with the control antibody immunoprecipitate. This result strongly suggests that p55alpha 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 p85alpha or -beta are replaced in p55alpha or - by unique 34-residue NH(2) 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(16) , paxillin(17) , hSOS1 (18) , p85 subunit of PI 3-kinase(19) ) is reported. Thus, the differences in the NH(2)-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 NH(2)terminal portions of the regulatory subunits of PI 3-kinase.


FOOTNOTES

*
This work was supported by a grant-in-aid for scientific research from the Japanese Ministry of Education. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D64045 [GenBank](rat p85alpha), D64046 [GenBank](rat p85beta), D64047 [GenBank](rat p55), and D64048 [GenBank](rat p55alpha).

§
To whom correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 3111); Fax: 81-3-5800-6798; asano-tky{at}umin.u-tokyo.ac.jp.

(^1)
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.


ACKNOWLEDGEMENTS

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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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.