Supplementary Materials and Methods

Plasmid construction

Tsc2 cDNAs for the rat mutants G1556S-type (GSM) and N525S-type (NSM) were generated by PCR. Briefly, the first PCR was performed with a High Fidelity PCR kit (Roche) using full-length rat Tsc2 cDNA (Kobayashi et al., 1997) as the template and the following primers (amino acid substitution sites are underlined): GSM 5’ fragment, RTSC22 (5’-ATTGAGCGGGCCATCTCCT-3’, forward, exon 33) and GSMR1 (5’-TAAGCCGACTCAGGCCTGT-3’, reverse, exon 36); GSM 3’ fragment, GSMF1 (5’-CTGACAGGCCTGAGTCGGCT-3’, forward, exon 36) and RTSC11 (5’-ATCCACAGAGGAAATGAGG-3’, reverse, exon 41); NSM 5’ fragment, NSMF1 (5’-GTCCCAATGAGGTGGTGTC-3’, forward, exon 10) and NSMR1 (5’-TCCAACAGACTGCTGAAGTG-3’, reverse, exon 14), NSM 3’ fragment, NSMF2 (5’-ACCCACCACTTCAGCAGTC-3’, forward, exon 14) and NSMR2 (5’-TAGCAGCAGGAAGTCAAAGG-3’, reverse, exon 17). The 5’ and 3’ GSM fragments amplified by the first PCR were mixed and subjected to a second PCR using the RTSC22/RTSC11 primer set; a similar procedure was carried out for the NSM fragments using the NSMF1/NSMR2 primer set. Amplified fragments from the second PCR were inserted into pCAG-FLAG-rTsc2-DEE (a wild-type rat Tsc2 cDNA expression vector) using the appropriate restriction enzymes to generate pCAG-FLAG-rTsc2-GSM and pCAG-FLAG-rTsc2-NSM (Cao et al., 2006). To generate the Y1571H-type mutant, two cDNA fragments covering the 5’-upstream and 3’-downstream regions from the mutation site were amplified using the following primers (the amino acid substitution site is underlined and the EcoRI site introduced for ligation without amino acid substitution is denoted with italics): 5’ fragment, RTSC57 (5’-ACATCTCTTACCAGTGCCAG-3’, forward, exon 26) and PSMR9 (5’-CAGGAATTCTGTGTGCCTATAAGAG-3’, reverse, exon 36); 3’ fragment, PSMF8 (5’-ACAGAATTCCTGACAGGCCTGGGT-3’, forward, exon 36) and STOP2-1 (5’-TGCTCGAGTCACACAAACTCTGTGAAGTC-3’, reverse, exon 41). These two fragments were used for sequential replacement of the normal sequences in pCAG-FLAG-rTsc2-DEE to obtain pCAG-FLAG-rTsc2-Ymut. For all mutant cDNA constructs, the accuracy of the amplified region was confirmed by sequencing. The GSM mutant cDNA clone was used to replace the normal sequences in pMGTsc2-WT (a wild-type Tg we have used in a previous study) to generate pMGTsc2-GSM. An N-terminally HA-tagged rat hamartin expression plasmid (pCAG-HA-rTsc1) was constructed using the full length rat Tsc1 cDNA (Satake et al., 1999) and the pCAG-GS expression vector (Niwa et al., 1991). The full-length rat Rheb cDNA was amplified by RT-PCR from embryonic total RNA with the primers RHEBF1 (5’-CCGAATTCCCTCAGTCCAAGTCCCGGA-3’) and RHEBR1 (5’-GGAGATCTTCACATCACCGAGCACGAAG-3’). After digestion with EcoRI and BglII, cDNA was subcloned into the modified pCAG-GS vector to generate the expression plasmid (pCAG-HA-rRheb) for the N-terminally HA-tagged Rheb.

Microinjection and generation of transgenic founders

Wistar female rats (Purchased from Charles River Laboratories, Kanagawa, Japan) were injected intraperitoneally with hormones to stimulate ovulation using the standard protocol (Hochi et al., 1992) and crossed with male Eker rats heterozygous for the Tsc2 mutation. Fertilized eggs at the pronuclear stage were collected from impregnated females. Tg DNAs were linearized with NotI, purified using a QIAquick kit (QIAGEN, Hilden, Germany), and microinjected into the fertilized eggs (Hochi et al., 1992). The eggs were then transferred into the oviducts of pseudo-pregnant Wistar females. All animal experiments were performed in accordance with a protocol approved by Juntendo University and PhoenixBio Inc.


Titles and Legends to Supplementary Figures

Figure S1. Laser-confocal imaging of transiently expressed tuberin variants in HEK293T cells. HEK293T cells expressing FLAG-tagged WT-tuberin (a), NSM-tuberin (b), GSM-tuberin (c), HA-tagged hamartin (d), FLAG-NSM-tuberin with HA-hamartin (e), or FLAG-tagged Y1571H-type tuberin with HA-hamartin (f). The methods for transfection and immunocytochemistry were essentially the same as described in the legend to Figure 1. The panels show the following images: upper left, DAPI (blue); upper right, anti-FLAG (tuberin, green); lower left, anti-HA (hamartin, red); lower right, merged image. Arrows indicate perinuclear-associated dense bodies (PNDBs).

Figure S2. Examination of phospho-peptides by mass spectrometry. Samples obtained from the immunoprecipitation with anti-FLAG were separated by SDS-PAGE. The tuberin bands were in-gel digested as previously described (Mineki et al., 2002). The tryptic peptides were extracted, the solvent was evaporated, and the peptides were redissolved in 10 µl of 1% formic acid. Mass spectrometry was performed using API-QSTAR pulsar i (Applied Biosystems, Framingham, CA) with a nanoliquid chromatograph (DiNa; KYA TECH Corporation, Tokyo, Japan) equipped with a 0.2 mm ID x 50 mm Magic C18 column. Amino acid sequences of the tryptic peptides were determined by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). (a) and (b) show representative LC-ESI-MS/MS spectra of peptides containing phosphorylated sites corresponding to human Ser939 and Ser1132, respectively. The doubly charged precursor ion had an m/z of 556.2 or 491.2, respectively. Phosphorylation status was investigated by comparing the peak intensity of phosphorylated to non-phosphorylated peptides in the LC-ESI-MS chromatogram (see Supplementary Table S1).

Figure S3. Generation of transgenic founder rats. (a) Schematic structure of the Tsc2 transgene. Horizontal lines indicate introns. Closed and open boxes indicate coding and non-coding regions, respectively. Hatched and striped boxes indicate the Tsc2 promoter region and the SV40 DNA fragment containing the poly-A additional signal, respectively. Restriction enzyme sites and the Rheb-GAP related domain are indicated above the diagram. The region used as the probe and the positions of translational initiation (ATG), termination (TGA), and the mutated site (GSM), are indicated below the diagram. The protocol for construction of GSM-Tg is described in the Supplementary materials and methods. (b) Screening of the founder rats. DNA samples (10 µg) were digested with BamHI and analyzed by Southern blotting with a 32P-labelled rat Tsc2 cDNA fragment, the 1.1 kb EcoRI-BamHI fragment covering exons 26-33 indicated in (a). A representative result for the GSM-Tg is shown. The band positions of the endogenous wild-type Tsc2 allele (Wild), the endogenous germ-line mutant Tsc2 allele of the Eker rat (Eker), and the Tg are indicated on the right. Size markers (kb) are shown on the left. (c) Northern blot analysis. Ten µg of total kidney RNA from the offspring of crosses of wild-type or transgenic rats were probed with the Tsc2 cDNA fragment or a fragment containing the SV40 poly-A additional signal. Representative results of a non-transgenic rat [(-)], a transgenic rat carrying WT-Tg (Wt) and a transgenic rat carrying GSM-Tg (GSM) with higher expression are shown.

Figure S4. Analysis of the characteristics of GSM-tuberin in vivo using fibroblasts from transgenically-rescued Eker homozygotes. (a) PCR genotyping of Tg and endogenous Tsc2 of control rats [(-) and +/Eker] and transgenically-rescued Tsc2Eker/Eker rats [(+) and Eker/Eker]. Genotyping was performed as described in the legend for Figure 4. (b) A hyper-rpS6 phosphorylation status found in cells from a Tsc2Eker/Eker rat rescued by GSM-Tg. Fibroblasts were obtained by digesting ear tissue from Tsc2Eker/Eker:WT-Tg (WT) or Tsc2Eker/Eker:GSM-Tg (GSM) rats using dispase-I (Sanko Junyaku, Tokyo, Japan) according to the manufacturer's recommended protocol, and maintained in DMEM supplemented with 10% FBS and antibiotics. Indirect immunofluorescent staining (IF) was performed using an anti-phospho(Ser 235/236)-S6 primary antibody and an Alexa Fluor 568-labeled anti-rabbit IgG secondary antibody. DAPI was used for nuclear staining. In the images, DAPI is shown as blue, and the phospho-rpS6 is stained red. Each image was acquired using the same configuration (e.g. same detector gain parameters, etc.). (c) Faster gel mobility shift and hypo-phosphorylation status at Ser939 of GSM-tuberin in cells from a Tsc2Eker/Eker rat rescued by GSM-Tg. Cell lysates from Tsc2Eker/Eker rats rescued by each Tg (WT or GSM) were analyzed by immunoblotting (IB) using anti-tuberin and anti-phospho(Ser939)-tuberin antibodies.


Supplementary References

Cao Y, Kamioka Y, Yokoi N, Kobayashi T, Hino O, Onodera M et al. (2006). Interaction of FoxO1 and TSC2 induces insulin resistance through activation of the mammalian target of rapamycin/p70 S6K pathway. J Biol Chem 281: 40242-40251.

Hochi S, Ninomiya T, Waga-Homma M, Sagara J, Yuki A. (1992). Secretion of bovine alpha-lactalbumin into the milk of transgenic rats. Mol Reprod Dev 33: 160-164.

Mineki R, Taka H, Fujimura T, Kikkawa M, Shindo N, Murayama K. (2002). In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Proteomics 2: 1672-1681.

Niwa H, Yamamura K, Miyazaki J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193-199.

Satake N, Kobayashi T, Kobayashi E, Izumi K, Hino O. (1999). Isolation and characterization of a rat homologue of the human tuberous sclerosis 1 gene (Tsc1) and analysis of its mutations in rat renal carcinomas. Cancer Res 59: 849-855.

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