7.a Mammalian two-hybrid assay
To investigate effects of lithium on the interaction between CREB and TORC1, TORC2, or TORC3 in a cellular system, a mammalian two-hybrid assay was employed. Here, only the interaction domains of the proteins of interest were used. HIT-T15 cells were transiently transfected with the luciferase-reporter gene G5E1B-Luc controlled by five repeats of the GAL4-binding site and with an expression construct encoding the CREB-bZip-wt fused to the constitutively active viral protein VP16, or VP16 alone. Cotransfected was an expression plasmid coding for the first 44 amino acids of TORC1, the first 53 amino acids of TORC2, or the first 46 amino acids of TORC3 fused to the DNA-binding domain of GAL4 (Figure 14, top). Through the GAL4-DBD GAL4-TORC11-44, GAL4-TORC21-53, or GAL4-TORC31-43 are tethered to the promoter of the G5E1B-Luc.
Transcription of the luciferase-gene is activated by VP16 if the VP16-bZip-wt interacts with a GAL4-fusion protein. Thus, luciferase activity is considered as a measure of interaction.
The results are shown in Figure 14. Compared to the control VP16, the basal activity of GAL4-TORC11-44 is increased 2.02-fold by VP16-bZip-wt (p<0.0001). VP16-bZip-wt enhanced the basal activity of GAL4-TORC21-53 3.31-fold (p<0.014) and the basal activity of GAL4-TORC31-43 2.03-fold (p<0.048). The treatment with 20 mM LiCl increased the luciferase activity mediated by GAL4-TORC11-44 6.8-fold to 1370.17 ± 108.84% (p<0.0001, Figure 14). Also for GAL4-TORC21-53 and for GAL4-TORC31-46 LiCl enhanced the luciferase activity, 4.8-fold to 1596.87 ± 260.97% (p<0.001) and 6-fold to 1225.41 ± 187.66% (p<0.001), respectively (Figure 14). The statistical analysis by two-way ANOVA confirmed significant effects of lithium on the interaction between VP16-bZip-wt and GAL4-TORC11-44, GAL4-TORC21-53, or GAL4-TORC31-46 with p<0.0001, but no difference was present between GAL4-TORC11-44, GAL-TORC21-53 and GAL4-TORC31-46 with respect to the extent of the effects of lithium.
Figure 14: Effects of lithium on the interaction between CREB and TORC1, TORC2, or TORC3 in the mammalian two-hybrid assay.
HIT-T15 cells were transiently transfected with the luciferase-reporter gene G5E1B under control of five repeats of the GAL4-binding site and an expression plasmid encoding the CREB basic leucine zipper (bZip) wild-type (wt) fused to the viral protein VP16, or VP16 alone. Cotransfected was an expression construct encoding the first 44 amino acids of TORC1, the first 53 amino acids of TORC2, or the first 46 amino acids of TORC3 fused to the DNA-binding domain of GAL4. A schematic illustration of the constructs is shown above the graph. The cells were treated with 20 mM LiCl for 30 h. Relative luciferase activity values are means ± SEM of three independent experiments performed in duplicate and are expressed in percent of the control VP16 for each TORC isoform. Compared to VP16 alone, the basal activity of GAL4-TORC11-44 was increased upon expression of VP16-bZip-wt. 20 mM LiCl strongly increased the luciferase activity. The expression of VP16-bZip-wt also increased the basal activity of GAL4-TORC21-53 and GAL4-TORC31-46. Comparable with GAL4-TORC11-44 treatment with 20 mM LiCl increased the basal activity of GAL4-TORC21-53 and GAL4-TORC31-46 significantly upon expression of VP16-bZip-wt.
Statistical analysis was performed by two-way ANOVA followed by Student’s t-test: ***p<0.001.
7.b In vitro GST pull-down assay
7.b.I Effects of lithium on the interaction between GST-CREB and full-length [35S]TORC1, [35S]TORC2, or [35S]TORC3
To examine the effect of lithium on the interaction between CREB and the full-length TORC1, TORC2, and TORC3 under cell-free conditions, GST pull-down assays were performed.
Bacterially expressed GST-CREB-wt immobilized on glutathione agarose (compare Figure 7B) and [35S]-labeled full-length TORC1, TORC2, and TORC3 (compare Figure 8) were employed with and without LiCl in a binding reaction. The amount of [35S]-labeled TORC recovered from GST-CREB-wt was determined by SDS-PAGE and subsequent autoradiography. A typical gel is shown below the graph (Figure 15). The control GST alone did not exhibit remarkable binding of [35S]TORC1, [35S]TORC2, or [35S]TORC3 with 12.03 ± 4.02% (p<0.001), 2.81 ± 0.40% (p<0.0001), and 20.53 ± 7.31% (p<0.002) of GST-CREB-wt, respectively (Figure 15), which was not changed by the presence of 20 mM LiCl. For GST-CREB-wt 20 mM LiCl increased the amount of [35S]TORC1 recovered from the sample to 175.84 ± 7.96% (p<0.006). In contrast, the amount of [35S]TORC2 or [35S]TORC3 recovered from GST-CREB-wt was not changed (Figure 15), as confirmed by two-way ANOVA.
Figure 15: Effects of lithium on the interaction between CREB and full-length TORC1, TORC2, or TORC3 in the GST pull-down assay.
GST-CREB wild-type (wt) and for control GST alone were expressed in E.coli, purified by affinity chromatography and immobilized on glutathione agarose. TORC1, TORC2, or TORC3 were labeled with [35S]methionine by in vitro transcription and translation. The amounts of [35S]-labeled proteins recovered from GST-CREB-wt or GST were analyzed by SDS-PAGE followed by autoradiography and densitometric analysis of the bands corresponding to [35S]TORC. Typical gels are shown below the graph. The data are mean values ± SEM of four independent experiments and are expressed in percent of GST-CREB-wt per group. Statistical analysis was performed by two-way ANOVA followed by two-sided paired Student’s t-test: **p<0.025.
GST alone exhibited no remarkable binding of [35S]TORC1, [35S]TORC2, or [35S]TORC3, compared to GST-CREB-wt. 20 mM LiCl increased the amount of [35S]TORC1 recovered from GST-CREB-wt.
The binding of [35S]TORC2 or [35S]TORC3 was not affected by the presence of 20 mM LiCl.
7.b.II Effects of lithium on the interaction between GST-CREB-wt and truncated [35S]TORC1327, [35S]TORC2347, or [35S]TORC3310
To examine a putative difference between full-length TORC proteins and shorter fragments for the effect of lithium on their interaction with GST-CREB-wt, truncated TORC proteins were employed in GST pull-down assays. Bacterially expressed GST-CREB-wt immobilized on glutathione agarose (compare Figure 7B) and [35S]-labeled TORC proteins comprising the first 327, 347, or 310 amino acids of TORC1, TORC2, or TORC3, respectively, were employed with and without LiCl in a binding reaction. The samples were analyzed by SDS-PAGE and autoradiography followed by densitometric analysis of the bands corresponding to the [35S]-labeled proteins. A typical gel is shown below the graph (Figure 16). The negative control GST alone did not exhibit remarkable binding of [35S]TORC1327, [35S]TORC2347, or [35S]TORC3310 with 5.07 ± 1.98% (p<0.0001), 1.85 ± 0.41% (p<0.0001), and 8.94 ± 2.70% (p<0.0001) of GST-CREB-wt, respectively (Figure 16), which was not altered by the presence of 20 mM LiCl. The amount of [35S]TORC1327 recovered from the GST-CREB-wt was increased by lithium to 213.19 ± 45.48% (p<0.05).
The presence of 20 mM LiCl did not change the amount of [35S]TORC2347 or [35S]TORC3310 recovered from GST-CREB-wt (Figure 16), as confirmed by two-way ANOVA.
Figure 16: Effects of lithium on the interaction between CREB and truncated TORC1327, TORC2347, or TORC3310 in the GST pull-down assay.
GST-CREB wild-type (wt) and, for control, GST alone were expressed in E.coli, purified by affinity chromatography and immobilized on glutathione agarose. TORC1327, TORC2347, or TORC3310
comprising the first 327, 347, or 310 amino acids of TORC1, TORC2, or TORC3, respectively, were labeled with [35S] by in vitro transcription and translation. The amounts of [35S]-labeled proteins recovered from GST-CREB-wt or GST were analyzed by SDS-PAGE followed by autoradiography and densitometric analysis of the bands corresponding to [35S]TORC. Typical gels are shown below the graph. The data are mean values ± SEM of four independent experiments and are expressed in percent on GST-CREB-wt per group. Statistical analysis was performed by two-way ANOVA followed by two-sided paired t-test: *p<0.05.
GST alone exhibited no remarkable binding of [35S]TORC1327, [35S]TORC2347, or [35S]TORC3310, compared to GST-CREB-wt. 20 mM LiCl increased the amount of [35S]TORC1327 recovered from GST-CREB-wt. The binding of [35S]TORC2347 or [35S]TORC3310 was not affected by the presence of 20 mM LiCl.