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DNA RESEARCH 4, 355-358 (1997) Short Communication Identification and Chromosome Assignment of a Human Gene Encoding a Novel Phosphatidylinositol-3 kinase Naohiko SEKI, 1 Yoshinori NIMURA, 2 Miki OHIRA, 1 Toshiyuki and Akira NAKAGAWARA2-* SAITO, 3 Shingo ICHIMIYA,2 Nobuo NOMURA, 1 Laboratory of Gene Structure 1, Kazusa DNA Research Institute, Yana 1532-3, Kisarazu, Chiba 292 Japan,1 Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuoh-ku, Chiba 260, Japan,2 and Genome Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263, Japan3 (Received 18 September 1997) Abstract We identified a novel phosphatidylinositol (PI) 3-kinase by screening human brain cDNA libraries with probes designed from the conserved kinase-domain sequence. Analysis of cDNAs indicated that two different forms of transcripts are present: one is the full-length form composed of 1,044 amino acid residues and the other is the short form that the N-terminal 216 amino acid residues including a putative p85 binding domain has been truncated (828 amino acid residues). Database search revealed the sequence of the full-length form to be identical to that recently registered by D. Chantry et al. (Accession No. U86453 in GenBank release, August 1997). Northern blot analysis showed this mRNA to be ubiquitously expressed in various tissues, with relatively higher expression was observed in spleen, thymus and leukocytes. Based onfluorescencein situ hybridization and PCR-based analyses with both human/rodent mono-chromosomal hybrid cell panels and radiation hybrid mapping panels, this gene was localized to chromosome region Ip36.2. This region is frequently lost in a variety of human malignancies, including neuroblastoma. The novel PI3K could be a candidate target of the Ip36 alteration that occurs in neuroendocrine tumors. Key words: PI3 kinase; Ip36.2; RT-PCR; neuroblastoma Phosphatidylinositol (PI) kinases are important regulatory molecules in eukaryotes and are involved in regulating cellular responses as diverse as vacuolar protein sorting, cytoskeletal organization, cell growth, and development.1"3 Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase and was initially identified through its association with viral oncoproteins and a number of growth factor receptors.4 A typical PI3K exists as a heterodimeric complex consisting of an 85-kDa (p85) regulatory subunit and a 110-kDa (pllO) catalytic subunit. 5 " 7 The 110-kDasubunit contains a C-terminal PI kinase domain, as well as a small domain of its N terminus that is sufficient for binding to the p85 subunit. The p85 subunit serves as an adapter and binds activated growth factor receptors and other tyrosine phosphorylated molecules through its two Src homology 2 (SH2) domains.8'9 Sequence analysis and characterization of the PI3Ks has made it possible to subdivide the PI3K superfamily into three classes. These classes are distinguishable not only on the basis of primary structure, but also by ~ ~—~ ; Gommunicated by Mituru lakanami * To whom correspondence should be addressed. Tel. 81-43-2645431, Fax. 81-43-265-4459, E-mail: [email protected]. or JP their in vitro substrate specificity and their likely mechanism of regulation and function in vivo.10'11 A number of genes have been identified that are similar to a large central portion of pi 10, but they differ from pi 10 at their N and C termini. Recently, several additional classes of PI3K have been identified, such as ATM, DNA-PKcs, FRAP and FRP1 that are involved in cell cycle regulation, checkpoint control and maintenance of genome integrity.12"17 To identify unknown superfamily members for better comprehension of the cell cycle regulation and signal transduction system, we carried out an reverse transcriptase-polymerase chain reaction (RT-PCR) method with degenerative primers, PI3Ks share a conserved C-terminal catalytic domain which is a specific amino acid stretch in the PI3K domain, To identify yet unidentified PI3Ks, we have designed degenerative PCR primers (5'-G-A-T/C-G-A-T/C-C-T-NC/A-G-N-C-A-A/G-G-A-3' and 5'-N-C-C-A/G-A-A-A/G-T-C-A/T/G-A-T-A/G-T-G-3') corresponding to the conserved amino acid sequences (D/E-D-L-R-Q-D/E and H-I-D-F-G) of the kinase domain, and performed an RTT-.^r. P C R 1S experiment with human brain mRNAs. 18 From the sequence analysis of the RT-PCR products, we obtained cDNA fragments for two independent novel PI kinases; 3§« [Vol. 4, N. Seki et al. Full-length form AAAAAAAA Truncated form AAAAAAAA Figure 1. Schematic representation of the two mRNA forms. The upper box indicates the product deduced from the full-length mRNA (Ace. No. U86453); the lower box shows that from the truncated mRNA (the clone HG1362 type, Ace. No. AB006753). The open boxes represent the common region shared between the two forms. The black box represents the PI3K domain. The hatched box shows the putative p85 binding domain. one is a PI kinase described previously,19 and the other is a new PI3K first described in the present study. To obtain the full-length cDNA clone, we searched a brain cDNA library of Kazusa DNA Research Institute enriched with relatively long cDNAs20 and clone HG1362 was found to contain the probed sequence. The entire sequence analysis of this clone by the shotgun strategy21 demonstrated that the clone contains a single open reading frame (ORF) composed of 828 amino acid residues. However, the sequence did not possess a putative p85 binding domain, suggesting that the 5'-moiety has been truncated (Ace. No. AB006753 in DDBJ/EMBL/GenBank). To obtain the full-length cDNA sequence, 5'-rapid amplification of cDNA ends (RACE)-PCR was performed using human brain and placenta cDNAs in the 5'-RACE-PCR kit of Clontech (USA). As a result, the two different forms of transcripts were identified: one is the form represented by clone HG1362 and the other form contains the extra 216 amino acids residues at its N terminus (Fig. 1). The region contains the p85 binding domain, as in the kinase pllO/3.22 Thus, we concluded that the complete form of our kinase is composed of 1,044 amino acid residues. However, the existence of the truncated form was also confirmed by RT-PCR using the mRNA fractions of various human tissues and primer sets specific for the 5' untranslated region (UTR) of the HG1362 clone (data not shown). We searched the latest DNA database and found that the sequence is identical to that recently registered by D. Chantry et al. (Ace. No. U86453 in Genbank data release, August 1997), except that asparagine at position 320 was serine in our sequence. By Northern blot analysis, we screened two human multiple tissue blots containing poly(A)+ mRNA for gene expression (Fig. 2). Its expression was ubiquitous although relatively higher expression was detected in spleen, thymus and leukocytes. Then chromosomal assignment of the novel PI3K gene was done by PCR analysis of human/rodent somatic cell hybrid panels and radiation hybrid panels. The PCR primer sets were designed for the 5'- 7.5 kb - 4.4 kb - 2.4 kb - (5-actin Figure 2. Northern blot analysis of the novel PI3K mRNA in various human tissues. Northern blot filters containing adult human poly(A) + RNAs (2 /jg/lane) were purchased from Clonetech Laboratories, Inc. (Palo Alto, CA), and hybridization and washing were performed following the manufacturer's instruction. The 3.5-kb cDNA fragment containing the entire open reading frame was labelled with [a-32P]dCTP and used as a hybridization probe. UTR of the gene. Specifically amplified PCR products with the oligonucleotide set were detected only from the hybrid containing human chromosome 1 (Fig. 3). We performed further mapping analysis using PCRbased radiation hybrid panels (Genebridge 4, Research Genetics, Inc.) and the same primer sets for the mono-chromosomal hybrid panels. Statistical analysis of the radiation hybrid data was performed using the RHMAPPER software package (http://wwwgenome.wi.mit.edu/cgi-bin/contig/rhmapper.pi). The data vector for the gene was 1000000000 0010001000 1011101000 0000000000 0000000000 1001000010 0000000000 0100011001 010 and the consequent report indicated that the gene was mapped between markers D1S253 and GCT15G02, both of which have been cytogenetically mapped on chromosome Ip36. This position is 1.82 cR proximal from D1S253. To confirm the PCR-based chromosome mapping by an independent approach, we performed R-bandingfluorescencein situ hybridization (FISH) using the PI phage DNA.23-24 The PI clone containing the cDNA sequence was isolated by the method described previously.25 The clear doublet signals were consistently demonstrated on the p36.2 region of chromosome 1. A typical FISH pattern is represented in Fig. 4 (a, b). Therefore, the gene was mapped on the p36.2 region of chromosome 1. Alteration and deletion of distal lp have been associated with several human tumors of neuroectodermal origin, including neuroblastoma, melanoma, and small cell lung carcinoma.26'27 No. 5] A Novel Phosphatidylinositol-3 kinase Gene 357 Figure 3. Chromosome mapping of the novel human PI3K gene. A PCR screening of human-rodent somatic cell hybrid panels was performed for mapping of the PI3K gene to human chromosome 1. DNA of the human-rodent somatic cell hybrid panels was purchased from the National Institute of General Medicine Service, Coriell Cell Repositories. Human, mouse and hamster genomic DNAs were also included as controls. Primers used for PCR amplification correspond to the 5' UTR region of HG1362 (5'-GCTCAACGTGGCAGGATAACC-3') and (5'- GTAGGGTCTGGGGAGTTCACA-3') (106-bp PCR product). PCR was carried out in a final volume of 10 /il containing 1 x LA-PCR buffer (Takara, Kyoto), 2 fiM each primer, 200 /xM each dNTP, 50 ng template DNA and 0.01 units of LA-Taq DNA polymerase (Takara, Kyoto). Temperature and time schedule were 30 cycles of 95°C for 20 sec. and 62°C for 1 min. Numbers on the top of each lane indicate the human chromosome contained in each somatic cell hybrid. Figure 4. Fluorescence in situ hybridization (FISH) of the PI3K gene. FISH was carried out using the PI phage clone harboring the PI3K gene as a biotinylated probe. Arrows indicate the signals on human chromosome Ip36.2. The metaphase spreads photographed with Nikon B-2A are shown in (a) and (b), and the position of FISH signals on the chromosome in diagram (c) are indicated by arrows. These cytogenetic changes could lead to either inactivation of tumor suppressor genes or activation of protooncogenes, contributing to the pathogenesis of these tumors. It is worth examining the alteration of the novel PI3K in these tumors. Acknowledgments: We thank K. Sato, K. Yamada and E. Suzuki for their technical assistance. This work was supported in part by a grant from Kazusa DNA Research Institute. References 1. Stephens, L. R., Jackson, T. R., and Hawkins, P. T. 1993, Agonist-stimulated synthesis of phosphatidylinositol (3,4,5)-trisphoshate: a new intercellular signalling system? Biochim. Biophys. Ada, 1179, 27-75. 2. Zakian, V. A. 1995, ATM-related genes: What do they tell us about functions of the human gene? Cell, 82, 685-687. 3. De Camilli, P., Emr, S. D., Mcpherson, P. D., Novick, P. 1996, Phosphoinositides as regulators in membrane traf- 358 N. Seki et al. fie, Science, 271, 1533-1539. 4. Carpenter, C. L. and Cantley, L. C. 1990, Phosphoinositide kinases, Biochemistry, 29, 11147-11156. 5. Otsu, M., Hiles, I., Gout, I. et al. 1991, Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-t/pp60c~src complexes, and PI3-kinase, Cell, 65, 91-104. 6. Fry. M., Hiles, I., Gout, I. 1992, Purification and characterization of phosphatidylinositol 3-kinase complex from bovine brain by using phosphopeptide affinity columns, Biochem. J., 288, 383-393. 7. Hiles, I. D., Otsu, M., Volinia, S. et al. 1992, Phosphatidylinositol 3-kinase: Structure and expression of the 110 kd catalytic subunit, Cell, 70, 419-429. 8. Hu, P., Margolis, B., Skolnik, E. Y., Lammers, R., Ullrich, A., and Schessinger, J. 1992, Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors, Mol. Cell. Biol, 12, 981-990. 9. McGlade, C. J., Ellis, C , Reedijk, M. et al. 1992, SH2 domains of p85a subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors, Mol. Cell. Biol, 12, 991-997. 10. Zvelebil, M. J., MacDougall, L., Leevers, S. et al. 1996, Structual and functional diversity of Phosphatidylinositol 3-kinase, Phil. Trans. R. Soc. Lond., 351, 217-23. 11. Leevers, S. J., Weinkove, D., MacDougall, L. K., Hafen, E., and Waterfield, M. D. 1996, The Drosophila phosphoinositide 3-kinase DpllO promotes cell growth, EMBO J., 15, 6584-6594.12. Brown, E. J., Albers, M. W., Shin, T. B. et al. 1994, A mammalian protein targeted by Gl-arresting rapamycinreceptor complex, Nature, 369, 756-758. 13. Sabatini, D. M., Erdjument-Bromage, H. et al. 1994, RAFT1: A mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs, Cell, 78, 35-43. 14. Zheng, X. F., Fiorentino, D., Chen, J., Crabtree, G. R., and Schreiber, S. L. 1995, TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin, Cell, 82, 121-130. 15. Weinert, T. A., Kiser, G. L., and Hartwell, L. H. 1994, Mitotic checkpoint genes in budding yeast and dependence of mitosis on DNA replication and repair, Genes Dev., 8, 652-665. 16. Hartley, K. O., Gell, D., Smith, G. C. M. et al. 1995, DNA-dependent protein kinase catalytic subunit: A [Vol. 4, relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product, Cell, 82, 849-856. 17. Kastan, M. B., Zhan, Q., El-Deiry, W. S. et al. 1992, A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia, Cell, 71, 587-597. 18. Saito, T., Seki, N., Matsuda, Y. et al. 1995, Identification of the human ERK gene as a putative receptor tyrosine kinase and its chromosomal localization to Ip36.1: A comparative mapping of human, mouse, and rat chromosomes, Genomics, 26, 382-384. 19. Saito, T., Seki, N., Ishii, H. et al. 1997, Complementary DNA cloning and chromosomal mapping of a novel phosphatidylinositol kinase gene, DNA Res., 4, 301-305. 20. Ohara, O., Nagase, T., Ishikawa, K.-I. et al. 1997, Construction and characterization of human brain cDNA libraries suitable for analysis of cDNA clones encoding relatively large proteins, DNA Res., 4, 53-59. 21. Nomura, N., Miyajima, N., Sazuka, T. et al. 1994, Prediction of the coding sequences of unidentified human genes. I. The coding sequences of 40 new genes (KIAA0001KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG1, DNA Res., 1, 27-35. 22. Hu, P., Mondino, A., Skolnik, E. Y., and Schlessinger, J. 1993, Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85, Mol. Cell. Biol., 13, 7677-7688. 23. Hori, T., Takahashi, E., Ayusawa, D., Takeishi, K., Kaneda, S., and Seno, T. 1990, Regional assignment of the human thymidylate synthase (TS) gene to chromosome band 18pll.32 by nonisotopic in situ hybridization, Hum. Genet, 85, 576-580. 24. Saito, T., Matsuda, Y., Ito, H., Fukaki, N., Hori, T., and Yamamoto, T. 1997, Localization of Zap70, the gene for a T cell-specific protein tyrosine kinase, to mouse and rat chromosomes by fluorescence in situ hybridization and molecular genetic linkage analyses, Mamm. Genome, 8, 45-46. 25. Ohira, M., Seki, N., Nagase, T. et al. 1997, Gene identification in 1.6-Mb region of the Down syndrome region on chromosome 21, Genome Research, 7, 47-58. 26. Schwab, M., Praml, C , and Amler, L. C. 1996, Genomic instability in lp and human malignancies, Gene Chromosomes Cancer, 16, 211-229. 27. Weith, A., Brodeur, G. M., Bruns, G. A. P. 1996, Report of the second international workshop on human chromosome 1 mapping 1995, Cytogenet. Cell Genet, 72, 113154.