QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online October 12, 2006
doi: 10.1242/10.1242/jcs.03199

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Patel, P. H. Articles by Tamanoi, F. Search for Related Content PubMed PubMed Citation Articles by Patel, P. H. Articles by Tamanoi, F. Social Bookmarking                         What's this?

Increased Rheb-TOR signaling enhances sensitivity of the whole organism to oxidative stress

¹ Molecular Biology Institute, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095-1489, USA
² Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095-1489, USA

View larger version (27K): [in a new window]   Fig. 1. Weak overexpression of Rheb stimulates TOR activity in adults. (A) Northern analysis of adult flies overexpressing Rheb with hs-GAL4 (hs>Rheb) and da-GAL4 (da>Rheb) shows increased Rheb transcript levels. (B) Western analysis of adults overexpressing Rheb with hs-GAL4 shows increased phosphorylation of T398 of S6K. Flies overexpressing Rheb at low levels develop into adult flies with normal body size and weight (wet) (P=0.3456, NS) (C) at a similar rate as control flies (P=0.3998, NS) (D). Error bars represent the s.d. Statistical comparison (T-test): all P values are based on comparison of the overexpressor with control (hsGAL4/w).

View larger version (14K): [in a new window]   Fig. 2. Mild overexpression of Rheb with hs-GAL4 (hs>Rheb) sensitizes adult flies to oxidative stress. Like control (hsGAL4/w) flies, hs>Rheb flies are not sensitive to daily feeding of 5% sucrose/PBS for 6 hours. However, hs>Rheb flies are sensitive to 5% H2O2 (mean survival is 33% after 5 days; n=166, P=0.0028) (A) as well as to another oxidative agent, paraquat (mean survival is 9% after 2 days; n=160, P=0.0006) (B). This mortality was not due to a general sensitivity of hs>Rheb flies to toxic compounds because these flies were found to be as sensitive to 25 mg/ml G418 as control flies (P=0.1193, NS). The mean survival on 25 mg/ml G418 is 64% for hsGAL4/w flies and 56% for hs>Rheb flies after 2 days (C). Error bars represent the s.d. Statistical comparison (T-test): all P values are based on comparison of the overexpressor with control.

View larger version (22K): [in a new window]   Fig. 3. Altering Rheb-TOR-S6K signaling can influence the response of adult flies to oxidative stress. Although increasing Rheb-TOR-S6K signaling sensitizes flies to oxidative stress (A), decreasing signaling through this pathway provides resistance to oxidative stress (B). Furthermore, Rheb-TOR signaling requires S6K to confer sensitivity to the whole organism to oxidative stress (C). The mean survival 24 hours after start of treatment with 5% H2O2 in 5% sucrose/PBS is 81% for hsGAL4/w flies (control) and 82% for daGAL4/w flies (control), but was only 33% for hs>TOR flies (n=117, P=0.0061) and 5% for da>S6KSTDETE flies (n=117, P=0.0014) (A). The mean survival 36 hours after start of treatment with 5% H2O2 in 5% sucrose/PBS is 57% for hs>Tsc1/2 flies (n=129, P=0.0169), 62% for da>Tsc2 flies (n=126, P=0.0059), 41% for da>TORFRB flies (n=106, P=0.3194), 97% for hs>S6KKQ flies (n=117, P=0.0030) and 84% for da>S6KKQ flies (n=117, P=0.0401), it was however, only 10% for hsGAL4/w flies (control) and 14% for daGAL4/w flies (control) (B). Overexpression of a dominant-negative form of TORFRB can partially rescue the sensitivity of flies overexpressing Rheb with hs-GAL4 (hs>Rheb) (C). Furthermore overexpression of a dominant-negative form of S6KKQ can fully rescue the sensitivity of flies overexpressing Rheb (P=0.1835, NS) (C). The mean survival after 6 days is 94% for hsGAL4/w flies (control), 17% for hs>Rheb flies, 40% for hs>Rheb; TORFRB flies and 75% for hs>Rheb; S6KKQ flies (C). Error bars represent the s.d. Statistical comparison (T-test): all P values are based on comparison of the overexpressor with controls (hsGAL4/w and daGAL4/w). (D) Scheme for the involvement of TSC-Rheb-TOR-S6K signaling in the stress response.

View larger version (27K): [in a new window]   Fig. 4. Increased Rheb-TOR signaling in muscle but not in neurons or fat bodies sensitizes adult flies to oxidative stress. (A) Overexpression of Rheb in neurons using the pan-neural driver, elav-GAL4 and the inducible (with RU486) GeneSwitch (GS) elav GS-GAL4 does not sensitize flies to oxidative stress. (B) In addition, overexpression of Rheb in the fat bodies with lsp2-GAL4 or DJ634-GAL4 does not sensitize flies to oxidative stress. Similarly, using the GeneSwitch S106 GS-GAL4, which allows expression in the abdominal fat body does not sensitize flies to oxidative stress. All values reported in A-C represent the mean survival of the indicated genotype 24 hours after exposure to 5% H2O2. (C) By contrast, flies overexpressing Rheb in muscle tissue (fed RU486) using the pan-muscle Gene Switch (GS) driver, myosin heavy chain (MHC) GS-GAL4 are sensitive to oxidative stress compared with control MHC GS>Rheb flies (not fed RU486). At 24 hours, the mean survival of MHC GS>Rheb flies not fed RU486 (n=102) is 90% whereas it is only 54% for MHC GS>Rheb flies fed RU486 (n=96, P=0.0084) (C). (D) A Kaplan-Meier survival plot further reveals the sensitivity of MHC GS>Rheb +RU flies to oxidative stress compared with MHC GS>Rheb –RU flies. Error bars represent the s.d. Statistical comparison (T-test): all P values are based on comparison of flies fed RU486 with those not fed RU486.

View larger version (12K): [in a new window]   Fig. 5. Altering Rheb-TOR-S6K signaling can influence the response of adult flies to starvation stress. (A) The mean survival 24 hours after the start of PBS starvation is 83% for hsGAL4/w flies and 98% for daGAL4/w flies, but only 12% for hs>Rheb flies (n=100, P=0.0339) and 5% for da>S6KSTDETE flies (n=100, P=0.0136). (B) The mean survival 36 hours after the start of PBS starvation is 66% for da>Tsc2 flies (n=110, P=0.0059) and 100% for hs>S6KKQ flies (n=117, P=0.0019), but only 20% for hsGAL4/w flies (control) and 23% for daGAL4/w flies (control). Error bars represent the s.d. Statistical comparison (T-test): all P values are based on comparison of the overexpressor with controls (hsGAL4/w and daGAL4/w).

View larger version (10K): [in a new window]   Fig. 6. Increased Rheb-TOR signaling through S6K promotes early senescence of negative geotaxis, the ability to travel against gravity. Although flies overexpressing Rheb (hs>Rheb) show no defect in this behavior 5 days after eclosion, hs>Rheb flies exhibit significant decrease in negative geotaxis 30 days post-eclosion (P=0.0002). Furthermore, co-overexpression of dominant-negative S6KKQ can rescue this defect in hs>Rheb flies. After 30 days, the mean percentage of flies traveling 5cm in 10 seconds is 93% for hsGAL4/w flies, 33% for hs>Rheb flies and 80% for hs> Rheb; S6KKQ flies (P=0.0705, NS). Error bars represent the s.d. Statistical comparison (T-test): P values are based on comparison of the overexpressor with the control (hsGAL4/w).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter