Key points are not available for this paper at this time.
Phosphatidylinositol 3,4,5-trisphosphate is a phospholipid signaling molecule involved in many cellular functions including growth factor receptor signaling, cytoskeletal organization, chemotaxis, apoptosis, and protein trafficking. Phosphorylation at the 3 position of the inositol ring is catalyzed by many different 3-kinases (classified as types IA, IB, II, and III), but the physiological roles played by each of the different 3-kinase isozymes during embryonic development and in homeostasis in animals is incompletely understood. Mammalian type IA kinase isozymes are heterodimers that are active at 37 °C when the catalytic 110-kDa subunit interacts through an amino-terminal binding domain with a regulatory 85- or 55-kDa subunit. Using gene targeting in embryonic stem cells, we deleted this binding domain in the gene encoding the α isoform of the 110-kDa catalytic subunit (Pik3ca) of the α isozyme of the type IA kinases, leading to loss of expression of the p110 catalytic subunit. We show that Pik3ca del/delembryos are developmentally delayed at embryonic day (E) 9.5 and die between E9.5 and E10.5. E9.5 Pik3ca del/delembryos have a profound proliferative defect but no increase in apoptosis. A proliferative defect is supported by the observation that fibroblasts from Pik3ca del/del embryos fail to replicate in Dulbecco's modified Eagle's medium and fetal calf serum, even with supplemental growth factors. Phosphatidylinositol 3,4,5-trisphosphate is a phospholipid signaling molecule involved in many cellular functions including growth factor receptor signaling, cytoskeletal organization, chemotaxis, apoptosis, and protein trafficking. Phosphorylation at the 3 position of the inositol ring is catalyzed by many different 3-kinases (classified as types IA, IB, II, and III), but the physiological roles played by each of the different 3-kinase isozymes during embryonic development and in homeostasis in animals is incompletely understood. Mammalian type IA kinase isozymes are heterodimers that are active at 37 °C when the catalytic 110-kDa subunit interacts through an amino-terminal binding domain with a regulatory 85- or 55-kDa subunit. Using gene targeting in embryonic stem cells, we deleted this binding domain in the gene encoding the α isoform of the 110-kDa catalytic subunit (Pik3ca) of the α isozyme of the type IA kinases, leading to loss of expression of the p110 catalytic subunit. We show that Pik3ca del/delembryos are developmentally delayed at embryonic day (E) 9.5 and die between E9.5 and E10.5. E9.5 Pik3ca del/delembryos have a profound proliferative defect but no increase in apoptosis. A proliferative defect is supported by the observation that fibroblasts from Pik3ca del/del embryos fail to replicate in Dulbecco's modified Eagle's medium and fetal calf serum, even with supplemental growth factors. Phosphorylated phosphoinositides generated by phosphoinositide (PI) 1The abbreviations used are: PI, phosphoinositide; PtdIns, phosphatidylinositol; ES, embryonic stems; En , embryonic day n ; kb, kilobase pair(s); PCR, polymerase chain reaction; bp, base pair(s); RT, reverse transcriptase; BrdUrd, bromodeoxyuridine; TUNEL, terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labeling. 3-kinases play crucial roles in signaling through receptors at the cell surface as well as in membrane trafficking mechanisms in Golgi and endosomal compartments, cytoskeletal organization, and regulation of apoptosis (1Batty I.H. Hickinson D.M. Downes C.P. Biochem. Soc. Trans. 1997; 25: 1132-1137Crossref PubMed Scopus (15) Google Scholar, 2Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (841) Google Scholar, 3Carpenter C.L. Cantley L.C. Biochim. Biophys. Acta. 1996; 1288: 11-16PubMed Google Scholar, 4Woscholski R. Dhand R. Fry M.J. Waterfield M.D. Parker P.J. J. Biol. Chem. 1994; 269: 25067-25072Abstract Full Text PDF PubMed Google Scholar, 5Kapeller R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (553) Google Scholar, 6Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 233-239Abstract Full Text PDF PubMed Scopus (835) Google Scholar, 7DeCamilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (661) Google Scholar, 8Hawkins P.T. Welch H. McGregor A. Eguinoa A. Gobert S. Krugmann S. Anderson K. Stokoe D. Stephens L. Biochem. Soc. Trans. 1997; 25: 1147-1151Crossref PubMed Scopus (31) Google Scholar, 9Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1323) Google Scholar). The genes for many PI 3-kinases have been isolated from a wide range of tissues and organisms. They are divided into three major classes based on their amino acid sequence, the homology among their lipid-kinase domains, and substrate specificities (6Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 233-239Abstract Full Text PDF PubMed Scopus (835) Google Scholar, 9Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1323) Google Scholar). Class I PI 3-kinases phosphorylate the 3′ sites of PtdIns, PtdIns 4-phosphate, and PtdIns 4,5-diphosphate to form PtdIns 3-phosphate, PtdIns 3,4-diphosphate, and PtdIns 3,4,5-trisphosphate. Class I PI 3-kinases can be further divided into two subclasses. The class IA PI 3-kinases, which include at least three isozymes, α, β, and δ, are heterodimers of a catalytic 110-kDa subunit (p110) and a regulatory subunit of 85 or 55 kDa (p85/p55) (10Domin J. Waterfield M.D. FEBS Lett. 1997; 410: 91-95Crossref PubMed Scopus (209) Google Scholar, 11Klippel A. Escobedo J.A. Hirano M. Williams L.T. Mol. Cell. Biol. 1994; 14: 2675-2685Crossref PubMed Scopus (127) Google Scholar, 12Vanhaesebroeck B. Welham M.J. Kotani K. Stein R. Warne P.H. Zvelebil M.J. Higashi K. Volinia S. Downward J. Waterfield M.D. Proc Natl Acad Sci U. S. A. 1997; 94: 4330-4335Crossref PubMed Scopus (374) Google Scholar, 13Inukai K. Anai M. Van Breda E. Hosaka T. Katagiri H. Funaki M. Fukushima Y. Ogihara T. Yazaki Y. Kikuchi O.Y. Asano T. J. Biol. Chem. 1996; 271: 5317-5320Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 14Fruman D.A. Cantley L.C. Carpenter C.L. Genomics. 1996; 37: 113-121Crossref PubMed Scopus (99) Google Scholar). The interaction of p85/p55 and p110 via the inter-Src homology 2 domain on p85/p55 and the amino-terminal 123 amino acids of p110 is critical for achieving maximal activity of this class of PI 3-kinases in mammalian cells at 37 °C (11Klippel A. Escobedo J.A. Hirano M. Williams L.T. Mol. Cell. Biol. 1994; 14: 2675-2685Crossref PubMed Scopus (127) Google Scholar, 15Dhand R. Hara K. Hiles I. Bax B. Gout I. Panayotou G. Fry M.J. Yonezawa K. Kasuga M. Waterfield M.D. EMBO J. 1994; 13: 511-521Crossref PubMed Scopus (295) Google Scholar, 16Yu J. Zhang Y. McIlroy J. Rordorf-Nikolic T. Orr G.A. Backer J.M. Mol. Cell. Biol. 1998; 18: 1379-1387Crossref PubMed Google Scholar). Despite a wealth of knowledge of the biochemistry and cell biological effects of phosphoinositides, very little information is available on the particular physiological roles played by these different enzymes and isozymes during mammalian development. Inhibitors of PI 3-kinases such as wortmannin (17Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1055) Google Scholar) and LY294002 (18Vlahos C.J. Matter W.F. Hui K.T. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar) are very useful for probing the functions of these enzymes but as with all inhibitor studies suffer from questions of specificity as to which enzymes and isozymes are being inhibited at the dosages used in a particular study. In contrast, specific mutations in genes encoding these enzymes would provide greater specificity. We have therefore begun a systematic program of generating mice carrying deletion alleles of the PI 3-kinases. Here we report the successful targeting of the α isoform of p110 to create a deletion allele that, in the homozygous state, leads to embryonic lethality at E9.5 due to a severe defect in the proliferative capacity of the embryo. 7.5 × 105 plaques from a 129SV mouse genomic library in the λFIX II vector (Stratagene) were screened (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 9.16-9.19Google Scholar) with probes derived from Pik3ca cDNA. Of three positive phage clones, one (F1) was selected for further study and subcloning, and an 8-kbSalI fragment was isolated and found to contain the first three coding exons of Pik3ca (see Fig. 1). A tetranucleotide repeat (CCTT) within an intron of Pik3ca was used for chromosome mapping using the Jackson Lab C57BL/6J and Mus spretusbackcross DNA panel map service. Two PCR primers flanking the tetranucleotide repeat (forward, GTA GGG ATG AAG GTG GAG AGG; backward, AGC TGA CTC AAT GGA AGT AGG G) were used for the amplification. PCR reaction was performed on 50 ng of DNA in a 20-μl volume containing 400 nm each primer, 200 nm of dNTP, 10 mm Tris-HCl (pH 8.3), 1.5 mm MgCl2, and 0.5 unit of Taq polymerase. Reactions were denatured at 94 °C for 5 min and then subjected to 30 step cycles consisting of 94 °C for 20 s, 57 °C for 60 s, and 72 °C for 30 s. PCR products were loaded on 3.5% MetaPhore agarose gel and separated at 70 V for 3 h. The targeting construct was made in the pPNT vector (20Tybulewicz V.L. Tremblay M.L. LaMarca M.E. Willemsen R. Stubblefield B.K. Winfield S. Zablocka B. Sidransky E. Martin B.M. Huang S.P. Ginns E. Nature. 1992; 357: 407-410Crossref PubMed Scopus (242) Google Scholar) using an 8-kb SalI fragment from phage F1 and resulting in replacement of the first coding exon with a neomycin resistance gene transcribed in reverse orientation from a mouse PGK promoter (see Fig.1). Electroporation into TC1 ES cells (21Deng C. Wynshaw-Boris A. Zhou F. Kuo A. Leder P. Cell. 1996; 84: 911-921Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar) and selection for homologous recombination were by published procedures. Recombinant ES cell lines were screened by PstI genomic Southern blot using probe B, located outside the targeted region of homologous recombination, and positive cell lines were confirmed by SacI genomic Southern blot using probe E (see Fig. 1). All studies described in this report were performed on mice carrying the mutant alleles on a 129/SV inbred background. Animal experiments were performed under protocols approved by the National Human Genome Research Institute's Animal Care and Use Committee under National Institutes of Health guidelines, “Using Animals in Intramural Research.” DNA was extracted from yolk sac (22Oakey R.J. Matteson P.G. Litwin S. Tilghman S.M. Nussbaum R.L. Genetics. 1995; 141: 667-674PubMed Google Scholar) and tails (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 9.16-9.19Google Scholar) by published methods. Two PCR reactions were performed for genotyping. One pair of primers derived from the exon deleted in the deletion allele (P110N, forward, CTC CCC AAT GGA ATG ATA GTG, and P110N, reverse, TCT TTT CTT CAC GGT TGC CT) amplifies a 260-bp fragment only from the wild type. The second pair of primers derived from the neomycin resistance gene (Neo1, 5′-AGA GGC TAT TCG GCT ATG ACT G, and Neo2, 3′-TTC GTC CAG ATC ATC CTG ATC) amplifies a 430-bp fragment only from the disrupted Pik3ca gene. PCR amplification was performed per the manufacturer's protocols (Life Technologies, Inc.). RNA extracted from 9.5-day-old embryos (22Oakey R.J. Matteson P.G. Litwin S. Tilghman S.M. Nussbaum R.L. Genetics. 1995; 141: 667-674PubMed Google Scholar) was used for reverse transcriptase (RT)-PCR per the manufacturer's protocols (Life Technologies, Inc.). Four pairs of primers were used for PCR amplification: P110N, forward, CTC CCC AAT GGA ATG ATA GTG, and reverse, TCT TTT CTT CAC GGT TGC CT, 260 bp; Neo1, 5′-AGA GGC TAT TCG GCT ATG ACT G, and Neo2, 3′-TTC GTC CAG ATC ATC CTG ATC, 431 bp; p110α, RT-1, sense primer, CTG TAT AAT GCT GGG GAG GAT GC, and RT-2, antisense primer, ATG CGG TAC AGG CCA GAG ATT C, 505 bp, amplify 825–1329 of p110α, sense primer, TCT GTC CCC TCT TGC and antisense primer, TAT ATC TCG CCC bp, amplify of cDNA. for amplification due to DNA primers were to and reactions were in Pik3ca and Pik3ca del/del E9.5 embryos were by on the day of cells were three with and the cell were on were in of on the were separated on and the were to membrane to the manufacturer's protocols were used for E. D. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar) with or in for at RT, and the was using and to the manufacturer's protocols were to for min at mice from were at day E9.5 with E9.5 embryos were separated from their yolk which were used for Pik3ca del/del were in at °C and then in were in with and in were to and in using and as described H. S. Full Text PDF PubMed Google Scholar). Pik3ca del/del E9.5 embryos were separated from their yolk which were used for genotyping. were in at °C then into and from the of embryos were by and and the of cells by Y. Y. J. Biol. 1992; PubMed Scopus Google Scholar). A tetranucleotide repeat within the intron of Pik3ca was when this fragment was to the The repeat was found to be between of C57BL/6J and M. A between these two by the Jackson DNA was used to map the to chromosome 3 at between and with no with and is with the region between and on the of chromosome 3 A (F1) isolated from a 129SV mouse genomic library (Stratagene) the first coding exons of Pik3ca The first coding exon of and the first of coding including the to The targeting construct of 3′ and 3 to the first coding recombination of the targeting construct in Fig. in deletion of this The of the deleted allele the as well as the domain that the binding to the inter-Src homology 2 domain of a regulatory subunit that is for catalytic activity of p110 in mammalian cells (11Klippel A. Escobedo J.A. Hirano M. Williams L.T. Mol. Cell. Biol. 1994; 14: 2675-2685Crossref PubMed Scopus (127) Google Scholar). The ATG at base of the second coding exon this ATG would the of the to be in within an M. J. Biol. 1993; Scopus Google Scholar) that a in position of ES the by genomic Southern blot 1). The ES cells were into to The the gene to the (F1) of mice from of and with del/del mice The del/del was 5 × the of Pik3ca mice the with a lethality in the were and no in growth or of from × of embryos or del/del embryos were found at but were and del/del embryos were found at but were and in a the of at which lethality is in Pik3ca del/del embryos at different of embryonic development were and of embryonic no del/del embryos were among of 60 E9.5 embryos and del/del as well as that be to be a of Pik3ca del/del a that be for by the the was different from the of with embryos these we that embryos to to die at and to E10.5. Pik3ca del/del embryos at E9.5 to be with and of E9.5 with and no defect in They with with the embryos from the The region of E9.5 Pik3ca del/del embryos was as with and was at and of the of del/del embryos embryos were in blot of RNA from wild type embryos and mouse tissues three and by a probe containing the coding region of Pik3ca The different from in of the gene and are the of of coding exons All three to be at from to of embryonic development and in different tissues of was in the expression of the different among in at E9.5 that the Pik3ca was the of RNA from Pik3ca Pik3ca del/del embryos that the deletion allele was in Pik3ca were in Pik3ca del/del when by of two of the deletion pair and and pair and the of the deleted by the targeted was del/del RNA pair and the neomycin resistance gene was from Pik3ca del/del The PI 3-kinases have been in signaling through growth P.T. Welch H. McGregor A. Eguinoa A. Gobert S. Krugmann S. Anderson K. Stokoe D. Stephens L. Biochem. Soc. Trans. 1997; 25: 1147-1151Crossref PubMed Scopus (31) Google Scholar, M.J. Waterfield M.D. Trans. R. Soc. 1993; PubMed Scopus Google S. M. Sci. U. S. A. 1994; PubMed Scopus Google Scholar) and in apoptosis through signaling through the kinase Cantley L.C. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, P. R. T. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). The delayed embryonic development and lethality del/del embryos and the of cells derived from these embryos to in be due to to or to apoptosis or We E9.5 Pik3ca del/del embryos for their to using in H. S. Full Text PDF PubMed Google Scholar) as well as for apoptosis by Y. Y. J. Biol. 1992; PubMed Scopus Google Scholar). In wild type the cells in three of an E9.5 the del/del embryos very little In to the severe proliferative no in apoptosis was The of cells apoptosis was and in and wild type embryos an of cells per del/del an of cells per The cells were located in and with a in of wild type del/del embryos at are from wild with and are from del/del with E of as in and C. in into by terminal was del/del embryos as with wild type that is no in apoptosis between wild type and Pik3ca from wild and embryos were by E9.5 embryonic fibroblasts from of wild type and Pik3ca In contrast, when were from del/del cells would to but be to in in Dulbecco's modified Eagle's medium with fetal calf and a of growth and used or in growth were were to a in signaling in cells by to Pik3ca S. M. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). The deletion in Pik3ca the p85/p55 binding but the containing the deletion be into a protein the first of the of was used for blot of fibroblasts from wild type and Pik3ca del/del this region of was deleted in the no protein was by two different of protein and from wild type and the of was in the with loss of expression of resulting from the targeting del/del embryos was when was used for of cells derived del/del embryonic fragment by in the wild type to be in blot of Pik3ca tissues by the were or in in del/del PtdIns 3,4,5-trisphosphate been in the to growth factor signaling through kinase receptors for growth growth factor factor growth growth and stem cell growth factor P.T. Welch H. McGregor A. Eguinoa A. Gobert S. Krugmann S. Anderson K. Stokoe D. Stephens L. Biochem. Soc. Trans. 1997; 25: 1147-1151Crossref PubMed Scopus (31) Google Scholar). at the 3 position of the inositol including PtdIns are generated by the type IA PI 3-kinases, which with in a B.M. P. S. Warne P.H. Downward J. Biol. 1997; Full Text Full Text PDF PubMed Google Scholar). In protein kinases, kinase and protein kinase are to be of PI 3-kinases by signaling (6Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 233-239Abstract Full Text PDF PubMed Scopus (835) Google Scholar). PI 3-kinases have been found to in protein trafficking P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (661) Google cytoskeletal P. Warne P.H. A. B.M. D. P. Waterfield M.D. A. Downward J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google and regulation of apoptosis Cantley L.C. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, P. R. T. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). that are many PI 3-kinases involved in many cellular is to to each of the isozymes of PI 3-kinase is or for carrying these many functions of PI 3-kinases. In is the α isozyme of PI 3-kinase for embryonic development or is a be for by such as the We mice homozygous for a deletion within Pik3ca by gene targeting and loss of expression of by using an a region of the protein involved in the deletion del/del have delayed development and between E9.5 and of embryonic development. The and growth were due to an of Pik3ca del/del embryos to in to with this severe defect was that all to cells from of del/del embryos from 129/SV inbred from Pik3ca del/del embryos to in and to the effects of fetal calf as well as growth such as and were with the of Siddle K. P.R. Biochem. J. 1996; PubMed Scopus Google PI 3-kinase is involved in signaling through but with the of S. M. Sci. U. S. A. 1994; PubMed Scopus Google that acid signaling PI 3-kinase to of the observation that PI 3-kinases the protein kinase and apoptosis in the Cantley L.C. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, P. R. T. Biol. 1998; Full Text Full Text PDF PubMed Google we were in was apoptosis in the Pik3ca in the In embryos of cells, in the region of the or We from these experiments that the PI 3-kinase by Pik3ca is for embryonic growth and the in Pik3ca del/del mice from an to in to growth factor signaling from apoptosis. we the that in phosphoinositides at the 3 position of inositol by the class IA PI 3-kinase we disrupted in these mice play a in the embryonic lethality through the wide of cellular in which PI 3-kinases are a of the del/del is that the embryos to to E9.5 of embryonic development. be an of PI 3-kinase that is or by E9.5 in of a increase in for PtdIns 3,4,5-trisphosphate that at this of development. a of in the only be to development. embryonic of PI 3-kinase with expression in development but PI 3-kinase catalytic such as the be to development to to The between E9.5 and is a of and as and in the R. The Scholar). A to and the to in to and We that as the for and del/del embryos at this the capacity of PI 3-kinase to for is to that the Pik3ca del/delembryos of of A in for by PI 3-kinase at the 3 position of inositol is supported by studies in which wortmannin inhibited in a membrane T. M. J. 1996; PubMed Scopus Google Scholar). One in the Pik3ca embryos was an increase in p85/p55 as by blot of at the that one of the of the Pik3ca allele on embryonic development be through p85/p55 loss of of of can with the binding of PI 3-kinases to by the binding between the homology 2 of the of the PI 3-kinases with their K. Y. Martin I. J.M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is very little information on the regulation of and of p85/p55 and an into the of this of p85/p55 is by to del/del cell The increase in protein in the Pik3ca del/del embryos to a of regulation that further We and Cantley for with and and for and We Williams for the and for We and for and We and for and with gene targeting and mouse was performed in the of Intramural National Genome Research National Institutes of
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