p60 Is an Adaptor for the Drosophila Phosphoinositide 3-Kinase, Dp110*

(Received for publication, February 27, 1997)

David Weinkove Dagger §, Sally J. Leevers Dagger , Lindsay K. MacDougall Dagger and Michael D. Waterfield Dagger par

From the Dagger  Ludwig Institute for Cancer Research, 91 Riding House Street, London W1P 8BT, United Kingdom and the § MRC Graduate Programme and the  Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The mammalian phosphoinositide 3-kinases (PI3Ks) p110alpha , beta , and delta  form heterodimers with Src homology 2 (SH2) domain-containing adaptors such as p85alpha or p55PIK. The two SH2 domains of these adaptors bind to phosphotyrosine residues (pY) found within the consensus sequence pYXXM. Here we show that a heterodimer of the Drosophila PI3K, Dp110, with an adaptor, p60, can be purified from S2 cells with a pYXXM phosphopeptide affinity matrix. Using amino acid sequence from the gel-purified protein, the gene encoding p60 was cloned and mapped to the genomic region 21B8-C1, and the exon/intron structure was determined. p60 contains two SH2 domains and an inter-SH2 domain but lacks the SH3 and breakpoint cluster region homology (BH) domains found in mammalian p85alpha and beta . Analysis of the sequence of p60 shows that the amino acids responsible for the SH2 domain binding specificity in mammalian p85alpha are conserved and predicts that the inter-SH2 domain has a coiled-coil structure. The Dp110·p60 complex was immunoprecipitated with p60-specific antisera and shown to possess both lipid and protein kinase activity. The complex was found in larvae, pupae, and adults, consistent with p60 functioning as the adaptor for Dp110 throughout the Drosophila life cycle.


INTRODUCTION

Studies in vertebrates, Drosophila melanogaster and Caenorhabditis elegans, suggest that both the structure and the function of receptor tyrosine kinases (RTKs)1 are highly conserved across metazoan organisms (1). Upon stimulation with an extracellular ligand, RTKs dimerize and transphosphorylate (2). This tyrosine phosphorylation enables the recruitment of signaling molecules containing SH2 or phosphotyrosine binding (PTB) domains that recognize phosphotyrosines within specific amino acid motifs (3). In this way, the SH2 domain-containing adaptors for Class IA PI3Ks (43) are recruited to tyrosine-phosphorylated RTKs and associated substrates containing the pYXXM motif (4). The recruitment of Class IA PI3Ks to activated RTKs coincides with a dramatic increase in the production of 3' phosphorylated phosphoinositides. These 3' phosphorylated phosphoinositides are thought to act as second messengers that affect cell growth, differentiation, membrane trafficking, and cytoskeletal organization (4).

In mammals, there are at least three Class IA PI3Ks, p110alpha , beta , and delta  (5, 6, 44) that can associate with a number of adaptors. Three distinct genes encode p85alpha , p85beta , and p55PIK, and additional adaptors are generated from alternatively spliced p85alpha transcripts (7-13). Each of these adaptors contains two SH2 domains, both of which selectively bind peptides containing phosphotyrosine with a methionine at the +3 position (pYXXM) (14, 15), and an inter-SH2 domain, which mediates binding to Class IA PI3Ks (16). The p85alpha and p85beta adaptors also contain an SH3 domain and a BH domain at the N terminus, whereas p55PIK and two splice variants of p85alpha have short N-terminal extensions. Despite this structural diversity, there has been no reported selectivity of binding between different adaptors and p110alpha , beta , or delta  (44).

We are using Drosophila to examine the role of the Class IA PI3Ks genetically and to provide an in vivo system to identify downstream targets. Many molecules downstream of RTKs in Drosophila are structurally and functionally homologous to their mammalian counterparts. The best characterized example of this is the Ras/MAPK pathway downstream of the Sevenless, Drosophila EGF receptor and Torso RTKs (17). Drosophila possesses a Class IA PI3K, Dp110 (also known as PI3K_92E), which is homologous to mammalian Class IA PI3Ks (18, 19). Previously, we have shown that the ectopic expression of Dp110 in larval imaginal discs affects cell growth but not cell differentiation (19).

Here we present the affinity purification of p60, the adaptor for Dp110, using immobilized phosphopeptides containing the pYXXM motif. Peptides derived from the purified protein were sequenced, and degenerate PCR and cDNA cloning were used to isolate the p60 cDNA. The structural and functional conservation of p60 with the mammalian adaptors is discussed. The Dp110·p60 complex possessed lipid and protein kinase activity and was found in Drosophila larvae, pupae, and adults and the S2 cell line. The genomic structure and location of the p60 gene has been determined to facilitate the identification and generation of genetic reagents to study the function of this PI3K adaptor in vivo.


EXPERIMENTAL PROCEDURES

Lysate Preparation and Affinity Purification

S2 cell lysates were prepared essentially as described (19) by lysis in buffer containing 1% Triton X-100 and the protease inhibitors, 1 mM phenylmethylsulfonyl fluoride, 18 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin, 5 mM benzamidine. Detergent lysates of Oregon R third instar larvae, pupae, and adult flies were prepared in the same buffer, but which additionally contained 5 µM diisopropylfluorophosphate and 15 µM N-alpha -p-tosyl-L-lysine chloromethyl ketone, by homogenization with a polytron (2 × 10 s, on ice). The homogenate was clarified by centrifugation at 20,000 × g for 1 h and then at 20,000 × g for 30 min. Affinity purification using phosphopeptide coupled to Actigel (Sterogene) beads was performed as described (19).

Peptide Microsequencing and cDNA Cloning

Proteins affinity purified from approximately 1010 S2 cells were resolved by SDS-PAGE and stained with Coomassie Blue R250. Each band was excised and digested with lysyl endopeptidase C (Wako Chemicals), and the resulting peptides were resolved by passage through an AX-300 pre-column and then a C18 (150 × 1 mm) column (Relasil). The peptides were sequenced at the 500 fmol level using an ABI Procise system. Peptides 6 and 9 (see Fig. 2A) were used to design the degenerate primers, CA(A/G)GA(A/G)(C/T)TITT(C/T)CA(C/T)TA(C/T)ATGGA and (C/G)(A/T)IACIGT(C/T)TC(A/G)TAIA(A/G)(C/T)TG(C/T)TG, respectively. Poly(A)+ mRNA was prepared from S2 cells using oligo(dT)-cellulose (Stratagene) and used to synthesize first strand cDNA with Moloney murine leukemia virus reverse transcriptase (Pharmacia Biotech Inc.). PCR reactions were performed with cDNA as a template and 0.025 units/µl Taq, 1.5 mM MgCl2, 0.2 mM dNTPs, and 1 µM each primer in a total volume of 50 µl. 40 cycles of amplification (94 °C for 30 s, 50 °C for 30 s, and 72 °C for 60 s) were performed. The 200-base pair product obtained was cloned into pGEM-T (Promega), sequenced, and found to encode peptides 7 and 8 (see Fig. 2A). This fragment was used to screen a lambda gt10 eye imaginal disc cDNA library (A. Cowan), and four positive clones were isolated that appeared to be identical by restriction mapping and by sequencing of the 5' and 3' ends. One clone was digested with EcoRI, and the two resulting fragments were subcloned into pBluescript SKII (Stratagene). PCR and direct sequencing of the original lambda clone showed the two fragments to be adjacent. Each fragment was sequenced in both directions with T3, T7, and p60-specific primers on an ABI 373 automated DNA sequencer.


Fig. 2. A, amino acid sequence of p60 aligned with the homologous regions of bovine p85alpha (9) and mouse p55PIK (10). The alignment was generated with the pileup algorithm of the Genetics Computer Group, Wisconsin (42). Identical amino acids in all three sequences are shaded. The SH2 domains are boxed. The nine peptide sequences obtained are underlined. Conserved hydrophobic residues in the inter-SH2 domains (possibly involved in coiled-coil structures) are marked with an asterisk. The phenylalanine and leucine residues implicated in the SH2 domain binding specificity are marked with a triangle (see "Results"). The proline-rich motif found in the mammalian adaptors is indicated as P2. B, comparison of the domain structure of p60 with mammalian adaptors for Class IA PI3Ks. The relative positions of the SH3, BH, SH2, and inter-SH2 domains are shown for p85alpha /beta . The proline rich motifs are indicated as P1 and P2. The C-terminal extension of p60 is shaded. C, genomic structure and localization of the gene encoding p60. The EcoRI restriction sites (E) are from the previously described map of the region (31). The distal breakpoint of Df(2L)al lies within the 3-kilobase EcoRI restriction fragment. The direction of transcription is from distal to proximal. The exons shown account for all the nucleotides within the cDNA clone. The first exon is bases 1-481, the second exon is bases 482-1368, and the third exon is bases 1369-3218. The initiation codon is at position 481, and the stop codon at position 1999. The coding region of the exons is shown in black.
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Immunological Methods

Peptides corresponding to the N terminus (CGGMQPSPLHYSTMRPQ, CGGSLVDPNEDELRMA) and the C terminus (CGGLYWKNNPLQVQMIQLQE, CGGSLEAEAAPASISPSNFSTSQ) of p60, including a CGG coupling linker at the N terminus, were coupled to maleimide-activated keyhole limpet hemocyanin as directed (Pierce). Antibodies were raised in rabbits against pools of N-terminal (alpha p60N) and C-terminal antigens (alpha p60C). Immunoblotting was performed using alpha Dp110 (1:1000) (19), alpha p60N (1:1000), or alpha p60C (1:2000) and developed with enhanced chemiluminescence as directed (Amersham Life Science). Immunoprecipitation was performed by incubating the lysate with a 1:200 dilution of antisera (6 µl) for 1 h at 4 °C and then adding protein A-Sepharose (Pharmacia) beads and incubating for an additional 30 min. The beads were washed in the same manner as the peptide-coupled beads (19).

Kinase Assays

For the lipid and protein kinase assays, the beads were washed three times in lysis buffer and twice in 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.1 mM EGTA. Lipid kinase assays were performed essentially as described (20) in 60 µl of 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.1 mM EGTA, 2.5 mM MgCl2, 100 µM ATP containing 2.5 µCi [gamma -32P]ATP and 200 µM sonicated phosphatidylinositol (Sigma). The reaction was incubated for 30 min at room temperature and terminated with acidified chloroform, and the lipid was extracted and resolved by thin layer chromatography with chloroform/methanol/4 M ammonium hydroxide (45:35:10). Protein kinase assays were performed in 30 µl of 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.1 mM EGTA, 2.5 mM MgCl2, 100 µM ATP containing 2.5 µCi [gamma -32P]ATP for 30 min at room temperature and resolved by SDS-PAGE on 7.5% polyacrylamide gels.


RESULTS

Affinity Purification of the Dp110·p60 Complex from Drosophila S2 Cells

Phosphopeptides, containing one or both of the pYXXM motifs found at positions 740 and 751 of the human PDGFbeta receptor, bind selectively to the adaptors for Class IA PI3Ks (14). When coupled to agarose beads, these phosphopeptides can be used to affinity purify heterodimeric complexes containing the adaptors bound to Class IA PI3Ks (9, 21). We investigated whether this approach could be used to identify an adaptor for Dp110, the Drosophila Class IA PI3K. Three proteins of approximately 145, 120, and 60 kDa were affinity purified from Drosophila S2 cells, using the tyrosine phosphorylated peptide GGYMDMSKDESVDpYVPML (pY751) coupled to agarose beads (Fig. 1). The same proteins were purified when the peptide was phosphorylated on tyrosine 740 or on both tyrosine 740 and tyrosine 751, but they were not recovered with beads lacking peptide (data not shown). The affinity purified complex possessed lipid and protein kinase activities, and immunoblotting with alpha Dp110 antisera showed that the 120-kDa protein was Dp110 (see below). We washed the complex at high stringency to determine which of the remaining bands was the adaptor for Dp110. Washing with lysis buffer containing increasing concentrations of sodium chloride removed the majority of the 145-kDa band, suggesting that p60 was the adaptor (Fig. 1). A large scale affinity purification was performed, and peptides derived from p60, p120, and p145 were sequenced (see "Experimental Procedures"). Three of the peptide sequences obtained from p120 confirmed that it was Dp110 (LMANYTGL, EYQVYGISTFN, and LHVLE). Two peptides sequenced from p145, FMEXIYTDVR and FXNNXXCGYIL, revealed that this protein was Drosophila phospholipase Cgamma (PLCgamma D) (22). Interestingly, human PLCgamma can be affinity purified from human cell lines using the same pYXXM phosphopeptide2, and the C-terminal SH2 domain of mammalian PLCgamma can interact with pYXXM motifs (14) though it binds preferentially to phosphotyrosines in other sequence contexts (15). Nine peptide sequences were obtained from p60 and used to design degenerate PCR primers. PCR amplification from first strand cDNA derived from S2 cell mRNA generated a 200-base pair fragment that was used to isolate p60 cDNAs from an eye imaginal disc cDNA library (see "Experimental Procedures"). These cDNAs contained an open reading frame that could encode a protein with a predicted size of 57.5 kDa, which contains all nine of the peptide sequences recovered from p60 (Fig. 2A).


Fig. 1. Affinity purification of p120 and p60 using the pY751 peptide implicates p60 as the adaptor for Dp110. Lysates from approximately 108 S2 cells were used for each sample. The final washes were carried out in buffer containing the indicated concentrations of NaCl, resolved by SDS -PAGE, and silver stained. p145 was removed at higher salt concentrations.
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Sequence Analysis of p60

Like the other identified adaptors for Class IA PI3Ks, the predicted amino acid sequence of p60 includes two SH2 domains and an inter-SH2 domain. However, the SH3 and BH domains in p85alpha and beta , the N-terminal extensions in p55PIK and the p55alpha splice variants, and the proline-rich SH3 domain-binding motifs (23) found in all mammalian adaptors are absent in p60 (Fig. 2B). p60 has a short N terminus (similar in size to the N terminus of p50alpha , a recently isolated splice variant of p85alpha (12)), and a unique C terminus of 70 amino acids that shows no significant similarity to other proteins. When the amino acid sequences of the core SH2-inter-SH2-SH2 region of p60, p85alpha , p85beta , and p55PIK are compared, p60 shows an equal degree of similarity to all three mammalian adaptors (Fig. 2A, data not shown).

The N-terminal and C-terminal SH2 domains are the most conserved regions of p60 and are 58% and 48% identical to the respective domains of bovine p85alpha . The three-dimensional structures of the N-terminal and C-terminal SH2 domains of p85alpha in complex with pYXXM phosphopeptides have been determined by x-ray crystallography and nuclear magnetic resonance (24, 25). These SH2 domain structures identify the amino acids responsible for the pYXXM binding specificity. These amino acids, including the phenylalanine of the beta strand E and the leucines of the loop between the alpha helix B and the beta strand G, are conserved in p60. Together, these three amino acids, shown in Fig. 2A, define the hydrophobic pocket that allows the specific binding of methionine three residues C-terminal to the phosphotyrosine (26).

The inter-SH2 domain of mammalian p85alpha mediates binding to the Class IA PI3K, p110alpha . Modelling studies of this inter-SH2 domain predict a two- or four-helix antiparallel coiled-coil structure similar to the solved crystal structure of the inter-SH2 domain of ZAP-70 (16, 27).3 The inter-SH2 domain of p60 is approximately 20% identical to the corresponding region of the mammalian adaptors. Despite this low homology, this region of p60 is likely to form a similar structure since it contains the leucine-rich heptad repeats characteristic of coiled-coil alpha helical bundles (see Fig. 2). Furthermore, BLITZ and BLASTP data base searches (28, 29) show that this region of p60 is significantly homologous to coiled-coil regions of proteins that form stable heterodimers (data not shown).

Genomic Location and Structure of the p60 Gene

A BLASTN (28) data base search with the p60 nucleotide sequence identified a sequence tagged site (STS, Dm0574) from the Berkeley Drosophila Genome Project that is identical to the 3'-untranslated region of the p60 cDNA and has been mapped to the genomic region, 21B6-C2 (30). To further characterize the genomic structure of the p60 gene, we performed Southern analysis of a lambda -phage contig of the region (kindly provided by M. Noll). This analysis determined the position and orientation of the p60 gene with respect to the published EcoRI restriction map (31). Furthermore, the exon/intron structure of the gene was determined by PCR, subcloning, and sequence analysis of the genomic clones (Fig. 2C). The gene encoding p60 has three exons and probably overlaps the breakpoint of the deficiency Df(2L)al at 21B8-C1.

Expression of p60 at Different Stages of the Drosophila Life Cycle

We initially purified p60 from S2 cells, an embryonically derived cell line. We next sought to characterize the expression of p60 at different stages of the Drosophila life cycle. The Dp110·p60 complex was affinity purified from Triton X-100 lysates of third instar larvae, pupae, and adult flies using pYXXM phosphopeptide beads (Fig. 3A). The complex is present in all stages examined although we consistently recovered lower levels from larvae than from the other stages. Immunoblotting with antisera against N- and C-terminal sequences of p60 and against Dp110 confirmed the presence of these proteins (Fig. 3B). An additional 55-kDa band can be seen that is immunoreactive with alpha p60N but not alpha p60C (Fig. 3, p60*). This protein might be a form of p60 that has been degraded from the C terminus, a splice variant of p60 lacking the C terminus or the product of another gene that cross-reacts with alpha p60N. We believe that this protein is a degraded form of p60 since its appearance coincides with lower levels of full-length p60 and because lysate preparation in the absence of certain protease inhibitors or following the freezing and thawing of samples resulted in increased levels of this smaller band (data not shown). In addition, the exon/intron structure of the p60 gene indicates that the 55-kDa band is unlikely to be encoded by an alternatively spliced transcript (Fig. 2C).


Fig. 3. Affinity purification of p60, Dp110, and PLCgamma from S2 cells, third instar larvae (L3), pupae, and adult flies. Lysates contained approximately 80 mg of protein. The proteins were resolved on SDS-PAGE and either silver stained (A) or transferred to nylon membranes and immunoblotted with alpha Dp110, alpha p60N and alpha p60C (B).
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Enzymatic Activity of the Dp110·p60 Complex

The Dp110·p60 complex possesses lipid and protein kinase activity when affinity purified from S2 cells with phosphopeptide beads. We confirmed these results using immunoprecipitation as an independent method of purifying Dp110·p60. Silver staining and immunoblots show that both alpha p60N and alpha p60C can immunoprecipitate the Dp110·p60 complex though alpha p60C is more efficient than alpha p60N (data not shown). Dp110, whether immunoprecipitated or affinity purified, is able to autophosphorylate and, to a lesser degree, to transphosphorylate p60 (Fig. 4A). The autophosphorylation is comparable to that shown by human p110delta (44), whereas the phosphorylation of p60 is reminiscent of the phosphorylation of p85alpha by mammalian p110alpha (32). However, p60 does not contain a phosphorylation site homologous to serine 608 in p85alpha (32) (Fig. 2A). Consistent with other Class IA PI3Ks in complex with their adaptors, both the immunoprecipitated Dp110·p60 complex and the affinity purified complex possessed lipid kinase activity as assessed by the conversion of phosphatidylinositol to phosphatidylinositol 3-phosphate (Fig. 4B).


Fig. 4. The Dp110·p60 complex possesses protein and lipid kinase activity. Immunoprecipitation and phosphopeptide purification (using DPY, the PDGFbeta receptor peptide doubly phosphorylated on tyrosines 740 and 751) were performed from equal volumes of S2 cell lysate with a 1:200 dilution of preimmune sera (alpha p60N and alpha p60C pooled), alpha p60N or alpha p60C. A, protein kinase assays were resolved by SDS-PAGE and autoradiographed. B, lipid kinase assays were performed using phosphatidylinositol as a substrate, resolved by thin layer chromatography, and autoradiographed.
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DISCUSSION

p60 has been affinity purified from Drosophila with a pYXXM phosphopeptide previously used to purify mammalian adaptors for Class IA PI3Ks. Analysis of the p60 amino acid sequence and the lipid and protein kinase activity of the Dp110·p60 complex indicates that p60 is both structurally and functionally homologous to the mammalian adaptors for Class IA PI3Ks. Since p60 is the most divergent member of the family identified to date, its sequence provides an insight into the evolution of the structure and function of these molecules. Notably, the residues responsible for the SH2 domain binding specificity are conserved, and the prediction of a coiled-coil structure for the inter-SH2 domains of the adaptor subunits for Class IA PI3Ks is supported.

Since we have shown that the pYXXM phosphopeptide can be used to purify adaptors in complex with Class IA PI3Ks from mammals and Drosophila, affinity purification with this phosphopeptide might also be used to isolate homologous PI3K complexes from many species. Interestingly, the SH2 domain of the Drosophila signaling molecule Drk also binds to the same phosphotyrosine motif recognized by its mammalian homologue Grb2 (33), Thus, affinity purification with peptides containing specific phosphotyrosine motifs might be used to isolate SH2 domain-containing proteins from various organisms.

Putative YXXM docking sites for the Dp110·p60 complex are found in the RTK Dret, the RTK substrate, Dos, and the Drosophila homologue of the insulin receptor, INR (34-36). However, it remains to be shown, either biochemically or genetically, whether these motifs are used in vivo. INR contains three YXXM motifs and is able to bind the N-terminal SH2 domain of mammalian p85alpha when phosphorylated (36). Class IA PI3Ks associate with the mammalian insulin receptor via multiple pYXXM motifs in its substrate IRS-1 and are thought to mediate many of the effects of insulin stimulation (37). Consistent with an analogous role for Class IA PI3Ks downstream of the Drosophila INR, certain mutations in inr and the ectopic expression of dominant negative Dp110 both affect imaginal disc cell growth (19, 38). It must be noted that mammalian adaptors for Class IA PI3K can also bind to the pYVXV motif in the Met receptor though with a lower affinity than for pYXXM motifs (39). Therefore, it is possible that p60 might recognize phosphotyrosine binding sites other than pYXXM.

It is likely that p60 is the only adaptor for Class IA PI3Ks present in Drosophila. However, we cannot rule out the possibility that additional adaptors exist. Together with Dp110, p60 was the predominant protein that was affinity purified from Drosophila with the pYXXM phosphopeptide (Fig. 3A). Immunoblotting of the affinity purified material with both alpha p60N and alpha p60C did not detect any larger splice variants of p60 that might contain SH3 or BH domains. Furthermore, we have not found exons encoding these domains when sequencing genomic DNA 9 kilobases upstream of the most 5'-exon of p60. Similarly, probing Northern blots with the p60 cDNA revealed only one band (data not shown). Thus, we conclude that any additional adaptors for Class IA PI3Ks that exist in Drosophila must be present at very low levels in the tissues that we have examined and/or other have a highly restricted expression pattern. Degenerate PCR and extensive cDNA library screening (18, 19, 40) have revealed only one member of each class of PI3K in Drosophila, suggesting that, in common with other gene families, there are less PI3K isoforms in Drosophila than in mammals.

If p60 is the only adaptor for Class IA PI3Ks in Drosophila, this implies that the adaptor of the common ancestor of vertebrates and flies consisted of the core SH2-inter-SH2-SH2 region. Even though SH3 domains are found in other Drosophila signaling molecules, for example, Drk (41), their absence in p60 suggests that the SH3 and BH domains found in mammalian p85alpha and beta  are the result of more recent evolution. p60 also lacks the SH3 domain-binding, proline-rich sequences found in all mammalian adaptors for Class IA PI3Ks. This again suggests that these motifs are involved in a more recently evolved mode of regulation that might be related to the presence of the SH3 domain found in p85alpha and beta .


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed: Ludwig Institute for Cancer Research, 91 Riding House St., London W1P 8BT, UK. Tel.: 44-171 878 4111; Fax: 44-171 878 4040; E-mail: mikew{at}ludwig.ucl.ac.uk. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number Y12498[GenBank].
1   The abbreviations used are: RTK, receptor tyrosine kinase; PI3K, phosphoinositide 3-kinase; pY, phosphotyrosine; SH2 and SH3, Src homology 2 and 3, respectively; BH, breakpoint cluster region homology; Class IA, the class of PI3Ks that associate with RTKs via SH2 domain-containing adaptors; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PLCgamma , phospholipase Cgamma ; contig, group of overlapping clones.
2   I. Gout, personal communication.
3   M. J. Zvelebil, personal communication

ACKNOWLEDGEMENTS

We thank Nick Totty, Alistair Sterling, Hans Hansen, and Justin Hsuan for peptide microsequencing and Marina Cotsiki and Christopher Odell for DNA sequencing. We are grateful to Yamanouchi Pharmaceutical Co. Ltd for phosphopeptides. We thank Joy Alcedo and Marcus Noll for providing genomic clones and restriction maps. We thank Thomas Twardzik, Thomas Raabe, and Martin Heisenberg for helpful discussions, and we are grateful to Ivan Gout, Markéta Zvelebil, Bart Vanhaesebroeck, Kyoichiro Higashi, and Ernst Hafen for useful discussions.


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