Fig 2: IP6increases solubility of mTOR and promotes a super-shift in its electrophoretic mobility.A and B, mTOR kinase reactions with or without IP6 (1 μM) and with or without MnCl2 (10 mM) were sampled over time and analyzed by western blot for soluble mTOR (A), followed by a final extraction of the insoluble material left in the tube after 90 min (B). Original western blot images are shown in Fig. S3A. In A, the fraction of soluble mTOR (soluble/total) over time of incubation was plotted. Dash line indicates the maximal possible amount of mTOR if completely in solution (0.25) given the volume collected (1/4). In B, the fraction of insoluble mTOR (insoluble/total) at the end of the reaction was plotted. In C and D, mTOR solubility (C) and kinase activity (D) were assayed with and without 0.1% CHAPS and with or without IP6 (10 μM) and samples collected at the end of 90 min for western blot of mTOR (C) or [32P]-peptide analysis (D). Original western blot images are shown in Fig. S3B. Shown are the scatter plots with mean and standard deviation of triplicate samples. (∗) indicates that IP6 changes are statistically significant as compared to equivalent control without IP6 using student’s t test (two tails, unpaired), with (A and B) (∗) p = 0.017; (∗∗∗) p < 0.0002; n.s = nonsignificant; (C and D) (∗∗∗)p < 0.0037; (#∗)p = 0.062; and (∗) p = 0.015. E, phosphorimager image of mTOR after autokinase reaction with [32P]-ATP, without (control) or with IP6 at the concentration indicated. F, western blots of mTOR after autokinase reaction with unlabeled ATP and without (control) or with IP6, as indicated. In lanes 4 and 5, IP6 was absent at the kinase reaction and added after SDS-loading buffer. In lanes 6 and 7, ATP and MnCl2 were not added to the kinase reaction, but only MnCl2 was shown to be necessary for the super-shift (see Fig. S4A). In E and F, no EDTA was used to stop the reactions. mTOR, mechanistic target of rapamycin; IP6, inositol hexakisphosphate.

Fig 3: IP6increases VMAXand decreases the apparent KMof mTOR for ATP, but does not affect mTOR/LST8/Raptor apparent KMfor ATP.A–B, Michaelis–Menten plots of initial velocity of mTOR without (open circles) or with 10 μM IP6 (closed squares) as a function of ATP concentration. In B, data were normalized by the measured relative levels of soluble mTOR at the end of the assay. The lines show the nonlinear regression curve fit, and calculated KM and VMAX are shown on Table 1. C, Michaelis–Menten plots of initial velocity of mTOR/LST8/Raptor without (open circles) or with 10 μM IP6 (closed squares) as a function of ATP concentration. Nonlinear regression curve fit showed a relative KM for ATP of 41.5 and 64 μM for reactions without or with IP6, respectively and VMAX of 1.11 x 105 and 1.09 x105 (counts/sec), respectively. D, product formed by mTOR/LST8/Raptor over prolonged incubation (16 h) as a function of ATP concentration. From nonlinear regression curve fit, we extrapolate that mTOR/LST8/Raptor required ATP at 56 μM (without IP6) and 64 μM (with IP6) for half of the maximal product formation and that the maximal product formed with IP6 was 1.54-fold higher than without IP6. Velocities were calculated from the plots shown in Fig. S5, C and D. mTOR, mechanistic target of rapamycin; IP6, inositol hexakisphosphate.

Fig 4: Inositol phosphates increase auto and peptide phosphorylation by mTOR in a concentration-dependent manner.A–F, mTOR (A, C, E, and F) or mTOR/LST8/Raptor (B and D) were incubated with [32P] γ-ATP in kinase reaction. (A and B) Autophosphorylation of mTOR and mTOR/LST8/Raptor with or without 100 μM of inositol or inositol phosphate species, inositol hexakis-sulfate (IS6), or glucose-6-phosphate (G6P), as shown by phosphoimager images of SDS-PAGE and quantification of the radiolabeled mTOR bands (shown below each band). Coomassie staining of the gels are shown on Fig. S1, A and B. C–F, peptide kinase assays in the presence of various concentrations of inositol phosphate species, as indicated. In C and D, data were normalized against the control (without inositol phosphates, dotted lines). Phosphorimager images of the spotted W3 papers for C and D are shown in Fig. S2, A and B. In F, peptide kinase reactions were done in the presence or absence of the phosphatase inhibitors, NaFl, ß-glycerophosphate, and nor-cantharidin. Data shown are the scattered plot with mean and standard deviations of quantified triplicate spots. (∗) indicate that the increase above control was statistically significant (p < 0.0001), using student’s t test (two-tails, unpaired). The p values for ANOVA analysis of each concentration group were (C) p = 2.677−13 (1 μM group); p = 7.1224−14 (10 μM group); p = 6.9552−12 (100 μM group) and for (D), p values were p = 8.233−17 (1 μM group); p = 1.0125−13 (10 μM group); and p = 1.3263−14 (100 μM group). mTOR, mechanistic target of rapamycin; NaFl, sodium fluoride.