FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse - PubMed (original) (raw)

. 2001 Nov 20;98(24):13796-801.

doi: 10.1073/pnas.241184198. Epub 2001 Nov 13.

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FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse

K E Hentges et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The FKBP-12-rapamycin associated protein (FRAP, also known as mTOR and RAFT-1) is a member of the phosphoinositide kinase related kinase family. FRAP has serine/threonine kinase activity and mediates the cellular response to mitogens through signaling to p70s6 kinase (p70(s6k)) and 4E-BP1, resulting in an increase in translation of subsets of cellular mRNAs. Translational up-regulation is blocked by inactivation of FRAP signaling by rapamycin, resulting in G(1) cell cycle arrest. Rapamycin is used as an immunosuppressant for kidney transplants and is currently under investigation as an antiproliferative agent in tumors because of its ability to block FRAP activity. Although the role of FRAP has been extensively studied in vitro, characterization of mammalian FRAP function in vivo has been limited to the immune system and tumor models. Here we report the identification of a loss-of-function mutation in the mouse FRAP gene, which illustrates a requirement for FRAP activity in embryonic development. Our studies also determined that rapamycin treatment of the early embryo results in a phenotype indistinguishable from the FRAP mutant, demonstrating that rapamycin has teratogenic activity.

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Figures

Figure 1

Figure 1

A mutation in the FRAP gene in flat-top mice and the predicted protein products. (A) Physical map of the flat-top region. BAC clones are shown in blue, PAC clones in orange. Genes identified in the flat-top interval from

blast

searches of clone end sequences are shown in boxes with the corresponding human PAC clone address in red. (B) RT-PCR by using primers that span the exon 7–8 splice junction produces products of 600 and 500 bp from flat-top RNA and one 500-bp product from wild-type RNA. flat, flat-top homozygote; wt, BTBR; neg, no template control. (C) The flat-top allele of the FRAP locus and the mRNAs it produces. The flat-top mutation is shown as a T to A base change in purple. The misspliced mRNA products are shown in red. mRNA A corresponds to the form that does not splice in this region, resulting in the inclusion of intron 7. Intron 7 has an in-frame stop codon (underlined nucleotides in intron) that would cause premature termination of translation. mRNA B is produced by an early splice in the intron, resulting in a 9-bp insert of intron sequence. Approximately 5% of the flat-top transcripts are wild type (wt). A similar proportion of the transcripts (mRNA C) splice into exon 8 resulting in a deletion of coding sequence. The genomic structure of the 5′ portion of the FRAP gene is shown in red, on the basis of the sequence of human PAC clone 647 M16 (GenBank accession no. AL049653). (D) Predicted protein products from the flat-top FRAP locus. The mRNA products shown in A are predicted to encode the proteins shown. mRNA A encodes an N-terminal fragment as a result of an in-frame stop codon in the intron. mRNA B encodes a three-amino acid insertion (in red in the one-letter amino acid code). mRNA C encodes a three-amino acid deletion (missing amino acids indicated by … ). (E) Western blot analysis of FRAP. An antibody recognizing the N terminus of the FRAP protein was used to probe a Western blot by using extracts from 293 cells, flat-top embryos, or wt control embryos. The position at which full length FRAP migrates is indicated by arrowheads. The blot has been overexposed in an attempt to detect the protein predicted by mRNA A. The expected mobility of this polypeptide is indicated by the arrow. The additional bands revealed by overexposure are caused by nonspecific binding by the antibody, as they are not blocked by an excess of soluble peptides used for the immunization that produced the antibody (data not shown). The identity of the FRAP band is known because it is competed by the peptide and it comigrates with a myc-tagged FRAP protein.

Figure 2

Figure 2

The flat-top mutation reduces FRAP activity significantly in homozygous embryos. (A) FRAP phosphorylation of p70s6k in vitro was analyzed by immunoprecipitation of FRAP from wild-type embryos (wt), flat-top mutant embryos (flat), and HEK293 cells, as described (16). There is ≈50% less labeled p70s6k substrate in flat-top embryos than in wild-type embryos or HEK293 controls. There was no activity in samples that were not incubated with substrate (−substrate). (B). Western blots showing the phosphorylation of p70s6k in flat-top mutant embryo extracts as compared with wild-type embryos and HEK293 cells. All of the signal represents S6 kinase. The slower migrating band in each lane is a splice variant of p70 generated by an alternative splice and sometimes designated p70a or p85. (Left) Anti-p70 antibody (Santa Cruz Biotechnology) was used to detect p70s6k on a Western blot. (Right Top) Santa Cruz p70s6k antibody on wild-type embryos, flat-top embryos, and 293 cells. (Center) NEB anti-p70-phospho-Thr-389 Western blot for the same samples as in Top. Note here that the flat-top embryos demonstrate little phosphorylation on Thr-389, a residue which is sensitive to FRAP activity. (Bottom) Double the amount of protein extracts was analyzed for Thr-389 phosphorylation. There is still little detectable Thr-389 phosophorylation in flat-top samples as compared with wild type.

Figure 3

Figure 3

FRAP flat-top mutants do not effect phosphorylation of 4E-BP1 at Ser-65 or Thr-70. 293T cells were cotransfected with pACTAG-2–4E-BP1 (HA-4E-BP1) and pcDNA3-flag-FRAP constructs. Cells were either treated with 100 ng/ml of rapamycin (+) or untreated (−). (A) Western blot analyses of whole cell extracts were performed with (Top) a phosphospecific antibody directed against 4E-BP1 serine 65 (α-P-Ser65), (Middle) an α-HA antibody to compare HA-4E-BP1 expression levels, and (Bottom) an α-flag antibody for detection of the flag-FRAP protein. All FRAP constructs were generated from a rapamycin-resistant form of the protein, Ser-2035-Thr. The experiment was carried out in duplicate with each of the transfected forms of FRAP. (B) The same cell extracts were used for Western blot analysis with (Top) a phosphospecific antibody directed against threonine 70 (α-P-thr-70); (Bottom) an α-HA antibody to examine HA-4E-BP1 expression. (C) The results of the serine 65 phosphorylation experiment were quantitated and expressed as a percent relative to the wild type without rapamycin treatment. The standard error is indicated.

Figure 4

Figure 4

FRAP and 4E-BP1 expression in wild-type embryos. (A) FRAP expression in a wild-type embryo at 8.5 dpc. The expression is most prominent in the midthoracic region of the embryo. (D) FRAP expression in a wild-type embryo at 9.5 dpc. The expression domain has expanded from that seen at 8.5 dpc. However, there appears to be little or no FRAP expression in the heart. Also, hindbrain rhombomeres 3 and 5 have higher levels of FRAP expression than surrounding tissues. (B) 4E-BP1 expression at 8.5 dpc. The expression is relatively low overall with the highest level of expression in the ventral portion of the forebrain and the surrounding mesenchyme. (E) Overall 4E-BP1 expression at 9.5 dpc. A section through the head (C) shows high levels of expression in the ventral telencephalic neuroectoderm and surrounding mesenchyme but not in the surface ectoderm.

Figure 5

Figure 5

Loss of FRAP activity does not affect regulation of cell size. Cell size comparisons of flat-top embryos and wild-type embryos. A single cell suspension was generated from the midthoracic region of either wild-type or flat-top mutant embryos at 9.5 dpc, and cells were measured to determine size. There is no significant difference between flat-top (Left) and wild-type (Right) cell diameters.

Figure 6

Figure 6

The flat-top phenotype and a rapamycin-induced phenocopy. (A) Wild-type (Left) and flat-top embryos at 9.5 days of gestation (dpc). The flat-top embryo is slightly developmentally delayed and smaller than somite matched wild-type embryos. Mutant embryos fail to turn, fail to form the telencephalic vesicles, and growth arrest by about 10.5 dpc. (B) Rapamycin treatment of pregnant females produces a phenocopy of the flat-top phenotype in wild-type embryos. Treatment of pregnant females with vehicle had no discernable effects on embryonic development.

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