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Science 336 (6080): 477-481

Copyright © 2012 by the American Association for the Advancement of Science

GSK3-TIP60-ULK1 Signaling Pathway Links Growth Factor Deprivation to Autophagy

Shu-Yong Lin1,*, Terytty Yang Li1,*, Qing Liu1, Cixiong Zhang1, Xiaotong Li1, Yan Chen1, Shi-Meng Zhang2, Guili Lian1, Qi Liu1, Ka Ruan1, Zhen Wang1, Chen-Song Zhang1, Kun-Yi Chien3, Jiawei Wu4, Qinxi Li1, Jiahuai Han1, and Sheng-Cai Lin1,{dagger}

1 State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Fujian 361005, China.
2 Beijing Institute of Radiation Medicine, Beijing 100850, China.
3 Molecular Medicine Research Center, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan.
4 School of Life Science, Tsinghua University, Beijing 100101, China.


Figure 1
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Fig. 1. Phosphorylation of TIP60 by two kinases in cells deprived of serum. (A) Differential electrophoresis mobilities among WT-TIP60 and TIP60 mutants. (B) Dephosphorylation of immunoprecipitated Myc-tagged WT and S86A but not S90A-TIP60 by calf-intestinal alkaline phosphatase (CIP). (C) In vitro phosphorylation of TIP60. In vitro kinase assays using His-tagged WT-TIP60 or S90A-TIP60 as substrates for immunoprecipitated Myc-cyclinB1/CDK1 complexes from human embryonic kidney (HEK) 293 T cells and purified bacterially expressed GST-GSK3β. (D) Time course of serum-deprived HCT116 cells. Endogenous TIP60 was immunoprecipitated with antibody against TIP60. Total cell lysates (TCL) and immunoprecipitates (IP) were immunoblotted as indicated. (E) Modification of endogenous TIP60. Lentivirus-mediated GSK3α and GSK3β double-knockdown HCT116 cells (GSK3-DKD) as well as LacZ siRNA-expressing control cells (ctrl) were placed in serum-free medium or standard medium for 12 hours. (F) HCT116 cells deprived of serum were treated with dimethyl sulfoxide (DMSO) (mock) or GSK3 inhibitors SB216763 or SB415286. (G) GSK3 activation increased phosphorylation of TIP60 on Ser86. HCT116 cells were placed in medium containing 20 μM inhibitor IV of AKT1 and AKT2 or 40 nM rapamycin for 16 hours. TCLs and TIP60 IPs were immunoblotted as indicated. Statistical analyses on the impacts of AKT inhibitor or rapamycin on GSK3β Ser9 phosphorylation were performed as described in the supplementary materials and are shown in fig. S5A.

 

Figure 2
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Fig. 2. TIP60 along with its Ser86 phosphorylation is essential in serum starvation–induced autophagy. (A) LC3 lipidation in TIP60 siRNA-expressing HCT116 cells (TIP60-KD) or the control cells (ctrl). Cells were deprived of serum for indicated times. Total proteins were extracted and immunoblotted as indicated. CQ, chloroquine. The relative amounts of LC3II were calculated from densitometry performed on immunoblots and normalized to the amount of tubulin. Data represent mean ± SEM of three independent experiments. Statistical significance was determined by analysis of variance (ANOVA); {dagger}P < 0.01, *P < 0.05, **P < 0.01 (ANOVA followed by Tukey). (B) LC3 lipidation in WT and TIP60S86A MEFs. Cytosolic proteins were then extracted and analyzed by means of immunoblotting. The graphs indicate p62 (degradation of which is a marker for autophagy activation)–to–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio and fold induction of LC3II. Data represent mean ± SEM of three independent experiments. {dagger}P < 0.01 (ANOVA), *P < 0.05, **P < 0.01 (ANOVA followed by Tukey); N.S., not significant. (C) TIP60 from heart homogenates isolated from 3 hours after birth or prenatal rat embryos was immunoprecipitated by using antibody to TIP60, followed by immunoblotting. Each lane represents a different individual. The relative Ser86 phosphorylation levels were calculated and normalized to total TIP60. Data are shown as mean ± SEM (n = 4 rats, each group). **P = 0.0034 (Student’s t test).

 

Figure 3
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Fig. 3. Acetylation of ULK1 by TIP60 upon serum deprivation. (A) Association of Myc-tagged TIP60 with cotransfected HA-tagged ATGs. Protein extracts were immunoprecipitated with antibody to HA 20 hours after transfection. (B) Increased association between TIP60 and ULK1 in cells deprived of serum. TIP60 immunoprecipitates from HCT116 cells serum starved for 16 hours were immunoblotted as indicated. Ratio of coimmunoprecipitated ULK1 to total ULK1 was calculated. Data represent mean ± SEM of three independent experiments. **P < 0.01 (Student’s t test). (C) Association of HA-ULK1 with Myc-TIP60. HEK293 T cells transfected with HA-ULK1 and Myc-TIP60 were treated with DMSO or 10 μM GSK3 inhibitor SB216763 for 12 hours. (D) Acetylation of endogenous ULK1. TIP60-KD HCT116 cells and ctrl cells were serum-starved for 12 hours. Acetylated proteins were immunoprecipitated with antibody to acetylated lysine. Ratio of acetylated ULK1 to total ULK1 was calculated. Data represent mean ± SEM of three independent experiments. N.S., not significant; **P < 0.01 (ANOVA followed by Tukey). (E) Inhibition of the acetylation of ULK1 by GSK3 inhibitor. HCT116 cells were pretreated with DMSO or 10 μM SB216763 for 4 hours before 12-hour serum starvation. Acetylated ULK1 was analyzed as in (D). (F) Phosphorylation-dependent activity of TIP60 toward ULK1. FLAG-TIP60 was immunoprecipitated from HEK293 T cells treated with DMSO or 10 μM SB216763 for 12 hours; TIP60 cotransfected with HA-GSK3β was used as a comparison. In vitro acetylation assays with purified His-ULK1 as a substrate were performed. Relative acetyltransferase activities of TIP60 toward ULK1 were calculated as the ratio of acetylated ULK1 to total ULK1. Data represent mean ± SEM of three independent experiments. **P < 0.01 (ANOVA followed by Tukey).

 

Figure 4
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Fig. 4. Requirement of TIP60-mediated acetylation of ULK1 in serum deprivation–induced autophagy. (A) In vitro acetylation assays were performed to determine TIP60 acetylation of immunoprecipitated HA-tagged WT-ULK1 or its lysine-to-arginine mutants. (B) In vitro coupled acetylation-phosphorylation assays were performed as described in the supplementary materials, materials and methods. WT-TIP60 or acetyltransferase-dead TIP60-DN was incubated with WT-ULK1 or 2KR-ULK1 in different combinations indicated. MBP was used as the substrate for ULK1 kinase in the presence of 32P-ATP. Data represent mean ± SEM of three independent experiments. N.S., not significant; **P < 0.01 (ANOVA followed by Tukey). (C) LC3 lipidation in ULK1–/– MEFs stably expressing WT-ULK1 or 2KR-ULK1. Cells were serum starved for 9 hours, and total cell lysates were analyzed for LC3 lipidation by means of immunoblotting. (D) Autophagosome formation in ULK1–/– MEFs. The cells were serum-starved for 9 hours after 24 hours of infection and were then fixed. LC3 positive puncta were shown as mean ± SEM of 5 random areas. N.S., not significant; **P < 0.01 (ANOVA followed by Tukey). (E) Comparison of LC3 lipidation in cells deprived of serum or glucose. HCT116 cells were pretreated with DMSO or GSK3 inhibitor SB216763 for 4 hours and then deprived of serum or glucose. Total proteins were analyzed for LC3 lipidation.

 


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