Tel2 binds newly synthesized PIKKs in an Hsp90-dependent manner. (A) Quantitative immunoblotting for Tel2, ATM, and mTOR in whole-cell lysates of the indicated cell lines using calibration with recombinant proteins as a standard. (B) Summary of the abundance of Tel2, ATM, and mTOR as determined in A. (C) Summary of Tel2-, Tti1-, and Tti2-associated proteins detected by mass spectrometry (see Supplemental Fig. 1). (D) Tel2 preferentially binds newly synthesized PIKKs. Immunoblots of Tel2-associated proteins in HeLa S3 cells treated with CHX (100 μg/mL) as indicated. Tel2 was isolated by Flag-HA tandem affinity purification, and the associated proteins were detected by immunoblotting as indicated. (E) Tel2 does not bind ATM phosphorylated on S1981. HeLa S3 cells expressing Flag-[HA]2-Tel2 were γ-irradiated (20 Gy) and lysed after 1 h. ATM was detected by immunoblotting using MAT3 (total ATM) or phospho-ATM Ab (ATM S1981-P) after Flag/HA affinity purification of Tel2. (F) Effect of the geldanamycin derivative 17-AAG (Gel) on Tel2–PIKK interactions. HeLa S3 cells were treated as in D, except that 0.5 μM 17-AAG was used to inhibit Hsp90 for the indicated times.

ATR and ATRIP complex formation depends on Tel2 and Hsp90 activity. (A) Immunoblots for ATR and Flag-ATRIP in cells with and without Tel2. (Last lane) Cells lacking Flag-ATRIP. (B, top panels) Immunoblots to detect total ATR and Flag-ATRIP, and autoradiography to detect ³⁵S-ATR in ATR IPs from cells treated as indicated. Cells were treated as in Figure 2A. The bar graph at the bottom shows the relative ³⁵S-ATR signal in the lanes above. (C) As in B, but using Flag IP for ATRIP. The top two panels are immunoblots for ATR and Flag-ATRIP, and the bottom panels represent detection of ³⁵S-labeled proteins. The bar graphs show the relative recovery of ³⁵S-labeled ATRIP and ATR in the ATRIP (Flag) IPs, determined based on the lanes shown above.

Direct binding of the conserved NTD patch to Tti1–Tti2 important for Tel2 function. (A) Molecular surface representation of scTel2 colored according to conservation among Tel2 orthologs (see Supplemental Fig. 2 for sequence alignment). (B) Close-up view of the conserved NTD patch shown in A. The highly conserved residues are colored red. (C) Binding of GST-scTel2 to Tti1–Tti2 complex. In vitro pull-down assay was carried out with purified recombinant proteins. Input (I), unbound (U), and bound (B) fractions were analyzed using a Coomassie-stained SDS-PAGE gel. The NTD but not CTD of scTel2 binds to the Tti1–Tti2 complex (cf. lanes 12 and 15). The L333Q/M345E mutant binds substantially lower amounts of the Tti1–Tti2 complex (cf. lanes 6 and 9). (D) Effect of Tel2 mutations on the stability of ATM, mTOR, and ATR. The indicated alleles of Tel2 were introduced into Tel2^F/− MEFs, and the endogenous Tel2 was deleted with 4-hydroxytamoxifen induction of Cre. At 120 h post-Cre, whole-cell lysates were analyzed by immunoblotting for the indicated proteins. (E) Effect of Tel2 mutations on the DNA damage response. The cells described in D were treated with IR (2 Gy) or UV (25 J/m²), and were processed for immunoblotting after 30 min and 1 h, respectively. The DNA damage response was monitored based on phosphorylation of Chk2 (top) and Chk1 (S345-P) (bottom). (F) Effect of Tel2 mutations on mTOR signaling after serum stimulation. (G) Summary of the phenotypes associated with Tel2 mutations. Levels of mTOR and ATM were determined as in D. The DNA damage response (DDR) was determined as in E. Proliferation (Prolif.) of cells treated with Cre was measured over 9 d.

Tel2 deletion affects formation of TORC1 and TORC2. (A) Schematic of the in vivo labeling experiments. (Top) Experiments evaluating the effect of Tel2 deletion. Tel2^F/− MEFs were treated for 6 h with 0.5 μM 4-hydroxytamoxifen to induce Cre and delete Tel2 (Tel2 KO) or were left untreated. Sixty hours later, the cells were labeled with ³⁵[S]-methionine and ³⁵[S]-cysteine for 1 h and washed with regular media before harvesting. (Bottom) Experiments evaluating the effect of 17-AAG (Gel) and rapamycin (Rap). Tel2-proficient MEFs were pretreated for 20 min with 0.1 μM rapamycin or 0.5 μM Hsp90 inhibitor 17-AAG and then labeled for 1 h as described above in the presence of the drugs. (B) Immunoblots for mTOR and Rictor in cells with and without Tel2. (C, top panels) Immunoblots to detect total mTOR and Rictor, and autoradiography to detect ³⁵S-mTOR in mTOR IPs from cells treated as indicated. The bar graph at the bottom shows the relative ³⁵S-mTOR signal in the lanes above; values represent averages, and the error bars represent the standard deviations derived from three independent experiments. (D) As in C, but using Rictor antibodies for the IPs. (E) Immunoblots for mTOR and Flag-Raptor in cells with and without Tel2. (F, top panels) Immunoblots to detect total mTOR and Raptor, and autoradiography to detect ³⁵S-mTOR in mTOR IPs from cells treated as indicated. (G) As in F, but using Flag IP for Raptor. The top two panels are immunoblots for mTOR and Raptor, and the bottom panels represent detection of ³⁵S-labeled proteins. Bar graphs show the relative recovery of ³⁵S-labeled mTOR and Raptor in the Raptor (Flag) IPs, determined based on the lanes shown above. (H) Immunoblots for mTOR and Flag-GβL in cells with and without Tel2. (I) Immunoblots to detect total mTOR, and autoradiography to detect ³⁵S-mTOR in mTOR IPs from cells treated as indicated. The bar graph at the bottom shows the relative ³⁵S-mTOR signal in the lanes above. (J) As in G, but using Flag IP for GβL. The top two panels are immunoblots for mTOR and GβL, and the bottom panels represent detection of ³⁵S-labeled proteins. The ³⁵S GβL signal overlaps with a weak nonspecific signal (asterisk). Bar graphs showing the relative recovery of ³⁵S-labeled mTOR and GβL in the GβL (Flag) IPs, determined based on the lanes shown above.

Overall structure of scTel2. (A) Schematic representation of Tel2. The domains discussed in the text, with their residue numbers for scTel2 and human Tel2 (HsTel2, in parentheses), are indicated, with the NTD colored cyan, the CTD colored pink, and the extended segment after the last NTD helical repeat in yellow. The two protease-sensitive flexible loops are marked with hatched lines. The crystallized scTel2 construct is shown as a line. (B) Overall view of scTel2, colored as in A. Dotted lines indicate disordered regions.