Figure 5. Loss of p53 decreases the distribution of TSC2 to and increases the level of Rheb in lysosomal membranes.

(A) Confocal microscopy of the colocalization of endogenous TSC2 and the lysosomal marker LAMP2. Endogenous TSC2 (green) and LAMP2 (red) were subjected to immunofluorescence labeling in wt and p53 null HCT116 cells. (B) Endogenous LAMP1 (green) and Rheb (red) were imaged in HCT116 cells as in (A). (C) Bar graphs show Manders’ colocalization coefficient for >50 cells expressed as a percentage ±SEM; the differences between wt and p53 null cells are significant for both comparisons (*p = 0.0001). (D) mTOR colocalized with LAMP2 in HCT116 cell wt or null for p53. Neither the degree of colocalization, nor the intensity of lysosomal mTOR differed between HCT cells with or without p53. (E) siRNA knockdown of p53 in H460 and A549 cells recapitulates the changes in lysosomal TSC2 and Rheb seen in HCT116 cells. TSC2 and Rheb colocalization with lysosomal markers LAMP1 and LAMP2 were quantitated as in panel C and are presented as means ± SEM (n=20). The differences between scrambled and siRNA for p53 were highly significant (P< 0.0001) for TSC2 distribution in both cell lines and for Rheb in H460 and at p=0.0004 for A549 and Rheb distribution. (F). Detection of Rheb in Raptor IPs from p53 null cells washed with low salt. Raptor IPs were washed with 100 mM salt prior to immunoblotting gels that were run long enough to separate IgG from Rheb. Note that the Rheb antibody cross-reacted with IgG but that a band corresponding to Rheb was found in p53 null cells but not p53 wt cell IPs that was absent in IgG IPs. Incubation of the Rheb antibody with its epitope peptide (1 μg/ml) blocked the detection of Rheb but not the IgG bands. The position of Rheb (red arrow) is marked by the location of the immunoreactive bands in lysates of HCT wt (+/+) and HCT p53 null (−/−) shown at the right. Blank indicates mixtures without lysate.