Plasma membrane translocation of REDD1 governed by GPCRs contributes to mTORC1 activation

Abstract

The mTORC1 kinase promotes cell growth in response to growth factors by activation of receptor tyrosine kinase. It is regulated by the cellular energy level and the availability of nutrients. mTORC1 activity is also inhibited by cellular stresses through overexpression of REDD1 (regulated in development and DNA damage responses). We report the identification of REDD1 in a fluorescent live-imaging screen aimed at discovering new proteins implicated in G-protein-coupled receptor signaling, based on translocation criteria. Using a sensitive and quantitative plasma membrane localization assay based on bioluminescent resonance energy transfer, we further show that a panel of endogenously expressed GPCRs, through a Ca 2+ /calmodulin pathway, triggers plasma membrane translocation of REDD1 but not of its homolog REDD2. REDD1 and REDD2 share a conserved mTORC1-inhibitory motif characterized at the functional and structural level and differ most in their N-termini. We show that the N-terminus of REDD1 and its mTORC1-inhibitory motif participate in the GPCR-evoked dynamic interaction of REDD1 with the plasma membrane. We further identify REDD1 as a novel effector in GPCR signaling. We show that fast activation of mTORC1 by GPCRs correlates with fast and maximal translocation of REDD1 to the plasma membrane. Overexpression of functional REDD1 leads to a reduction of mTORC1 activation by GPCRs. By contrast, depletion of endogenous REDD1 protein unleashes mTORC1 activity. Thus, translocation to the plasma membrane appears to be an inactivation mechanism of REDD1 by GPCRs, which probably act by sequestering its functional mTORC1-inhibitory motif that is necessary for plasma membrane targeting.

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways

“…The mechanism of REDD1-YFP translocation has been confirmed and characterized in our previous study (15), demonstrating that REDD1 is a novel link between GPCR and the mammalian Target Of Rapamycin Complex 1 mTORC1 kinase signaling that regulates translation.…”

“…This is probably because of the low abundance of this ubiquitously expressed protein and also because our previous study on REDD1 has shown that its main phosphorylated residues (ser19, thr23, and thr25) were not necessary for REDD1 translocation in response to GPCR activation (15). Low abundant proteins and the absence of triggered phosphorylations are two obvious drawbacks of the phosphoproteomic approaches for analyzing new proteins of the GPCR signaling pathways.…”

“…Low abundant proteins and the absence of triggered phosphorylations are two obvious drawbacks of the phosphoproteomic approaches for analyzing new proteins of the GPCR signaling pathways. In our previous study, the translocation of REDD1 was further characterized using a quantitative assay based on Bioluminescence Resonance Energy Transfer performed in living cells that permits quantification of dynamic interactions of proteins with the plasma membrane (15). Using this technique, we could show that REDD1 is specifically translocated to the plasma membrane in response to activation of five out of six endogenously expressed GPCRs that were tested, namely, those that couple preferentially to Gq, Gs or Gi heterotrimeric G proteins, but not to G12.…”

“…Previously, we reported the identification and characterization of one of these translocated proteins, REDD1, as a novel target of GPCR signaling (15). The data are compared with the results of phospho-proteome investigations of signaling pathways governed by GPCRs (16 -22).…”

Interleukin-6 influences stress-signalling by reducing the expression of the mTOR-inhibitor REDD1 in a STAT3-dependent manner

“…Also LPS induces REDD1 expression both in short and long term experiments [58]. The complexity of crosstalk scenarios including REDD1 becomes even more apparent when taking into account that REDD1 activity is not only controlled by regulation of REDD1 expression but also by sequestering REDD1 to certain cellular compartments [59]. These observations indicate that REDD1 expression is regulated in composite crosstalk scenarios that will be subject to future investigations.…”