Heat shock protein 90 regulates phosphatidylinositol 3-kinase-related protein kinase family proteins together with the RUVBL1/2 and Tel2-containing co-factor complex

Abstract

Heat shock protein 90 (Hsp90), a conserved molecular chaperone for a specific set of proteins critical for signal transduction including several oncogenic proteins, has been recognized as a promising target for anticancer therapy. Hsp90 inhibition also sensitizes cancer cells to DNA damage. However, the underlying mechanisms are not fully understood. Here, we provide evidence that Hsp90 is a general regulator of phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins, central regulators of stress responses including DNA damage. Inhibition of Hsp90 causes a reduction of all PIKK and suppresses PIKK-mediated signaling. In addition, Hsp90 forms complexes with RUVBL1/2 complex and Tel2 complex, both of which have been shown to interact with all PIKK and control their abundance and functions. These results suggest that Hsp90 can form multiple complexes with the RUVBL1/2 complex and Tel2 complex and function in the regulation of PIKK, providing additional rationale for the effectiveness of Hsp90 inhibition for anticancer therapy, including sensitization to DNA damage.

Figure 1

Inhibition of heat shock protein 90 (Hsp90) activity decreases the abundance of all phosphatidylinositol 3‐kinase‐related protein kinase (PIKK) proteins and the downstream signaling. (A–F) HeLa TetOff cells were treated with vehicle or 2 μM 17‐allylamino‐17‐desmethoxygeldanamycin (17‐AAG) for 12 or 24 h, then the cells were untreated, treated with 10 Gy IR, or 100 J/m² of UV, and incubated for 1 h. Total cell lysates were analyzed by western blotting with the indicated antibodies. To estimate the protein abundance, 33 and 11% of the 17‐AAG, IR and UV‐untreated samples were loaded. The anti‐P‐S/TQ antibody recognizes phosphorylated serine or threonine in the SQ motif, potential phosphorylation sites by ATM/ATR/SMG‐1/DNA‐PKcs (E, lower panel). Asterisks indicate some examples of phosphoproteins affected by the 17‐AAG treatment (E, lower panel). ATM, ataxia telangiectasia mutated; ATR, ATM‐and Rad3‐related; DNA‐PKcs, DNA‐dependent protein kinase catalytic subunit; mTOR, mammalian target of rapamycin; SMG‐1, suppressor with morphological effect on genitalia 1; TRRAP, transformation/transcription domain‐associated protein.

Figure 2

Heat shock protein 90 (Hsp90) interacts with two other phosphatidylinositol 3‐kinase‐related protein kinase (PIKK) regulators, RUVBL1/2 and Tel2 and their associated proteins. (A) RUVBL1 interacted with Hsp90, a Hsp90 co‐factor (NOP17), URI complex and Tel2 complex. Tet‐inducible streptavidin‐binding peptide (SBP)‐tagged RUVBL1 stable HEK 293 cells or control cells, which express tag peptides only, were treated with 1 ng/mL doxycycline for 3 days. Cytoplasmic cell extracts were affinity purified with streptavidin sepharose, and biotin‐eluted fractions were analyzed by western blotting with the indicated antibodies. (B,C) Protein interactions of Tel2, SMG‐10, Tti2 and URI. HeLa TetOff cells were immunoprecipitated with anti‐Tti2, anti‐Tel2 antiserum or normal rabbit serum (NRS) (B), or anti‐SMG‐10, URI, or normal rabbit IgG (NRIgG) (C). The immunoprecipitates were analyzed by western blotting with the indicated antibodies. Input: 1, 0.33, 0.11 and 0.037% (B) or 0.5, 0.17 and 0.06% (C) of the amount immunoprecipitated. (D) RPAP3 interacts with all PIKK. HeLa TetOff cells were transfected with pcDNA5/FRT/TO/NTAP‐GST, pcDNA5/FRT/TO/NTAP‐NOP17, pcDNA5/FRT/TO/NTAP‐RPAP3 or pcDNA5/FRT/TO/NTAP‐SMG‐10. The cell extracts were subjected to affinity purification with streptavidin sepharose 36 h later and analyzed by western blotting with indicated antibodies. ATM, ataxia telangiectasia mutated; ATR, ATM‐and Rad3‐related; DNA‐PKcs, DNA‐dependent protein kinase catalytic subunit; mTOR, mammalian target of rapamycin; NOP17, nucleolar protein 17; RPB5, RNA polymerase II subunit 5; SMG‐1, suppressor with morphological effect on genitalia 1; TRRAP, transformation/transcription domain‐associated protein.

Figure 3

Knockdown of RUVBL1/2 and Tel2 interacting proteins partially affects the phosphatidylinositol 3‐kinase‐related protein kinase (PIKK) abundance. (A,C,D) HeLa TetOff cells were transfected with the indicated siRNA. Sixty hours after transfection, total cell lysates were analyzed by western blotting with the indicated antibodies. Same knockdown samples were used in panels A, C, D in Figure 3, and panels A and B in Figure 4. Mean values of the relative protein levels compared to the nonsilencing (NS) control from two independent experiments are shown under the blots. (B) HeLa TetOff cells were treated with vehicle or 2 μM 17‐AAG for 12 h and total cell lysates were analyzed by western blotting with the indicated antibodies. To estimate the protein abundance, 33 and 11% of the NS sample (A,C,D) or untreated sample (B) were loaded. ATM, ataxia telangiectasia mutated; ATR, ATM‐and Rad3‐related; DNA‐PKcs, DNA‐dependent protein kinase catalytic subunit; mTOR, mammalian target of rapamycin; SMG‐1, suppressor with morphological effect on genitalia 1; TRRAP, transformation/transcription domain‐associated protein.

Figure 4

Knockdown of RUVBL1/2 and Tel2 interacting proteins affects the phosphatidylinositol 3‐kinase‐related protein kinase (PIKK) signaling. (A,B) HeLa TetOff cells were transfected with the indicated siRNA. Sixty hours after transfection, total cell lysates were analyzed by western blotting with the indicated antibodies. Same knockdown samples were used in panels A,C,D in Figure 3, and panels A and B in Figure 4. To estimate protein abundances, 33 and 11% of the nonsilencing (NS) control were loaded. (C,D) HeLa TetOff cells were transfected with the indicated siRNA. Sixty hours later, cells were untreated, treated with 100 J/m² of UV (C), or 10 Gy IR (D), and incubated for 1 h. Total cell lysates were analyzed by western blotting with the indicated antibodies. The anti‐P‐S/TQ antibody recognizes phosphorylated serine or threonine in the SQ motif, potential phosphorylation sites by ATM/ATR/SMG‐1/DNA‐PKcs (B,D). Relative phosphorylation levels of p70S6K (A), Upf1 (B), Chk‐1 (C), Chk‐2 (D) were indicated under each blot. ATM, ataxia telangiectasia mutated; ATR, ATM‐and Rad3‐related; DNA‐PKcs, DNA‐dependent protein kinase catalytic subunit; SMG‐1, suppressor with morphological effect on genitalia 1.

Figure 5

Knockdown of DNA‐PKcs or mTOR reduces the ATM abundance. HeLa TetOff cells were transfected with two independent siRNA targeted to each phosphatidylinositol 3‐kinase‐related protein kinase (PIKK). Sixty hours after the transfections, total cell lysates were analyzed by western blotting with the indicated antibodies. To estimate the PIKK abundance, 33 and 11% of the nonsilencing (NS) control sample were loaded. The relative protein levels of each PIKK compared with the NS control levels were graphed. The values are an average of the data from two siRNA from two independent experiments. ATM, ataxia telangiectasia mutated; ATR, ATM‐and Rad3‐related; DNA‐PKcs, DNA‐dependent protein kinase catalytic subunit; mTOR, mammalian target of rapamycin; SMG‐1, suppressor with morphological effect on genitalia 1; TRRAP, transformation/transcription domain‐associated protein.