Eccentric contractions increase the phosphorylation of tuberous sclerosis complex-2 (TSC2) and alter the targeting of TSC2 and the mechanistic target of rapamycin to the lysosome - PubMed (original) (raw)

Eccentric contractions increase the phosphorylation of tuberous sclerosis complex-2 (TSC2) and alter the targeting of TSC2 and the mechanistic target of rapamycin to the lysosome

Brittany L Jacobs et al. J Physiol. 2013.

Abstract

The goal of this study was to determine whether the mechanical activation of mechanistic target of rapamycin (mTOR) signalling is associated with changes in phosphorylation of tuberous sclerosis complex-2 (TSC2) and targeting of mTOR and TSC2 to the lysosome. As a source of mechanical stimulation, mouse skeletal muscles were subjected to eccentric contractions (ECs). The results demonstrated that ECs induced hyper-phosphorylation of TSC2 and at least part of this increase occurred on residue(s) that fall within RxRxxS/T consensus motif(s). Furthermore, in control muscles, we found that both mTOR and TSC2 are highly enriched at the lysosome. Intriguingly, ECs enhanced the lysosomal association of mTOR and almost completely abolished the lysosomal association of TSC2. Based on these results, we developed a new model that could potentially explain how mechanical stimuli activate mTOR signalling. Furthermore, this is the first study to reveal that the activation of mTOR is associated with the translocation of TSC2 away from the lysosome. Since a large number of signalling pathways rely on TSC2 to control mTOR signalling, our results have potentially revealed a fundamental mechanism via which not only mechanical, but also various other types of stimuli, control mTOR signalling.

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Figures

Figure 1

Figure 1. Eccentric contractions induce an increase in TSC2 phosphorylation

Mouse TA muscles were collected 1 h after a bout of eccentric contractions (EC) or the control condition. A, samples were subjected to Western blot (WB) analysis with the indicated antibodies. B, samples were immunoprecipitated with a TSC2 antibody and then treated with, or without, λ phosphatase (λPPase) and subjected to WB analysis for total TSC2. C, samples were immunoprecipitated with a TSC2 antibody, or a non-immune (IgG) antibody as a negative control, and then subjected to WB analysis for RxRxxS*/T* consensus motif phosphorylation. All images are representative of the average results obtained from _n_= 3–5 per group. _n_= number of samples.

Figure 2

Figure 2. Eccentric contractions induce translocation of TSC2

Mouse TA muscles were collected 1 h after a bout of eccentric contractions (EC) or the control condition. A, samples were separated into cytosolic (C) and crude membrane (M) fractions, and then subjected to Western blot analysis for TSC2, mTOR and markers of cytosolic (LDH) and membrane (Na+,K+-ATPase)- associated proteins. B, the amount of TSC2 and mTOR in the membrane fraction was divided by the total amount found in both fractions, and then this value was expressed as a percentage of the mean value obtained in the control samples. Values in the graph represent the mean + SEM, _n_= 8 per group. *Significantly different from control, _P_≤ 0.05.

Figure 3

Figure 3. Eccentric contractions induce dissociation of TSC2 from the lysosome

C2C12 myoblasts were transfected with RNAi constructs that express GFP and a scrambled (control) RNAi (A and B), or GFP and an RNAi targeting TSC2 (C and D). At 72 h post transfection, the myoblasts were subjected to IHC for GFP and TSC2. A and C, merge of the signals obtained for GFP and TSC2; B and D, greyscale images of the signal obtained for TSC2 in A and C, respectively. E–J, semi-ultrathin sections from control (E–G) or eccentrically contracted (EC; H–J) TA muscles were subjected to IHC for laminin (LN), LAMP2 and TSC2. E and H, merge of the signals obtained for LN, LAMP2 and TSC2. F and I, greyscale image of the signal obtained for LAMP2 in E and H, respectively. G and J, greyscale image of the signal obtained for TSC2 in E and H, respectively. Pullouts to the right of each image represent a more highly magnified region. Scale bars represent 10 μm in the full size images and 2 μm in the pullouts. K and L, frequency scatterplots were generated by comparing the intensity of the signal for LAMP2 versus the intensity of the signal for TSC2 within every pixel of the images from control (K) or EC (L) muscles. The pink dashed lines represent the position of the threshold values that were used to characterize pixels as intensely positive for LAMP2 or TSC2. M, the number of pixels in each image that were intensely positive for both LAMP2 and TSC2 (i.e. colocalized) were quantified and then expressed as a percentage of the mean value obtained in the control samples. K–M, the data in each group were acquired from 23 randomly selected images (>3 × 107 pixels) that were obtained from 4 independent muscles. Values in the graph represent the mean + SEM. *Significantly different from control, _P_≤ 0.05.

Figure 4

Figure 4. Eccentric contractions enhance the association of mTOR with the lysosome

C2C12 myoblasts were transfected with RNAi constructs that express GFP and a scrambled (control) RNAi (A and B), or GFP and an RNAi targeting mTOR (C and D). At 72 h post transfection, the myoblasts were subjected to IHC for GFP and mTOR. A and C, merge of the signals obtained for GFP and mTOR; B and D, greyscale images of the signal obtained for mTOR in A and C, respectively. E–J, semi-ultrathin sections from control (E–G) or eccentrically contracted (EC; H–J) TA muscles were subjected to IHC for laminin (LN), LAMP2 and mTOR. E and H, merge of the signals obtained for LN, LAMP2 and mTOR. F and I, greyscale image of the signal obtained for LAMP2 in E and H, respectively. G and J, greyscale image of the signal obtained for mTOR in E and H, respectively. Pullouts to the right of each image represent a more highly magnified region. Scale bars represent 10 μm in the full size images and 2 μm in the pullouts. K–L, frequency scatterplots were generated by comparing the intensity of the signal for LAMP2 versus the intensity of the signal for mTOR within every pixel of the images from control (K) or EC (L) muscles. The pink dashed lines represent the position of the threshold values that were used to characterize pixels as intensely positive for LAMP2 or mTOR. M, the number of pixels in each image that were intensely positive for both LAMP2 and mTOR (i.e. colocalized) were quantified and then expressed as a percentage of the mean value obtained in the control samples. K–M, the data in each group were acquired from 34 randomly selected images (>4 × 107 pixels) that were obtained from 4 independent muscles. Values in the graph represent the mean + SEM. *Significantly different from control, _P_≤ 0.05.

Figure 5

Figure 5. Conceptual model of how mechanical stimuli activate mTOR signalling

In this model, the skeletal muscle lysosomes serve as a major regulatory centre for controlling mTOR signalling. In response to the mechanically induced signalling events (shown with arrows), mTOR signalling transitions to its active state. See Discussion for details.

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