mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β

Abstract

Glycogen synthase kinase 3β (GSK3β) phosphorylates and thereby regulates a wide range of protein substrates involved in diverse cellular functions. Some GSK3β substrates, such as c-Myc and Snail, are nuclear transcription factors, suggesting the possibility that GSK3β function is controlled through its nuclear localization. Here, using ARPE-19 and MDA-MB-231 human cell lines, we found that inhibition of mTOR complex 1 (mTORC1) leads to partial redistribution of GSK3β from the cytosol to the nucleus and to a GSK3β-dependent reduction of the levels of both c-Myc and Snail. mTORC1 is known to be controlled by metabolic cues, such as by AMP-activated protein kinase (AMPK) or amino acid abundance, and we observed here that AMPK activation or amino acid deprivation promotes GSK3β nuclear localization in an mTORC1-dependent manner. GSK3β was detected on several distinct endomembrane compartments, including lysosomes. Consistently, disruption of late endosomes/lysosomes through a perturbation of RAS oncogene family member 7 (Rab7) resulted in loss of GSK3β from lysosomes and in enhanced GSK3β nuclear localization as well as GSK3β-dependent reduction of c-Myc levels. These findings indicate that the nuclear localization and function of GSK3β is suppressed by mTORC1 and suggest a link between metabolic conditions sensed by mTORC1 and GSK3β-dependent regulation of transcriptional networks controlling cellular biomass production.

Targeted enhancement of flotillin-dependent endocytosis augments cellular uptake and impact of cytotoxic drugs

“…DHHC5 localizes at the postsynaptic membrane of neurons through phosphorylation of Y533 in a manner regulated by Fyn 35 . To test the possible regulation of DHHC5 by Fyn and/or phosphorylation upon USMB treatment, we first examined the phosphorylation of DHHC5 using phos-tag gel electrophoresis, a method that exaggerates the apparent molecular weight differences elicited by protein phosphorylation 52 . We observed that USMB and USMB+desipramine elicited a reduction in the apparent molecular weight of These results indicate that USMB treatment leads to a reduction of DHHC5 phosphorylation, and that this phenomenon is controlled by Fyn.…”

“…Endogenous flotillin-1 or -2, DHHC5 or γH2AX were labelled by performing indirect immunofluorescence as previously described 52 . Samples were fixed in a solution of 4% PFA and permeabilized by Triton-X100 (for flotillin labeling experiments) or ice-cold methanol (for DHHC5 labeling experiments).…”

“…Quantification of whole-cell intensity of flotillin-1, DHHC5, or internalized fluid-phase A488dextran, or nuclear γH2AX was performed as previously described 51,52 using ImageJ software (National Institutes of Health, Bethesda, MD) 58 . Detection and analysis of diffraction-limited internalized flotillin-1 puncta (Figure 1G-H Figure 2B-D) was performed as previously described for cell surface clathrin structures 49,62 .…”

“…Whole cell lysates were prepared in Laemmli sample buffer, resolved by SDS-PAGE and probed with specific antibodies, and the resulting signal intensity quantification were performed as previously described 52 . To examine phosphorylation of DHHC5 (Figure 4A), we used the phostag gel system, which results in exaggeration of differences in apparent molecular weight of phosphorylated forms of specific proteins, as previously described 52 .…”

“…Statistical analysis was performed as previously described 52 . Measurements of samples involving one experimental parameter and more than two conditions (Figures 2A-B, 2H, S4A, S4C) were analyzed by one-way ANOVA, followed by Tukey post-test to compare differences between conditions, with p < 0.05 as a threshold for statistically significant difference between conditions.…”