WAC Regulates mTOR Activity by Acting as an Adaptor for the TTT and Pontin/Reptin Complexes

Abstract

SUMMARY The ability to sense energy status is crucial in the regulation of metabolism via the mechanistic Target of Rapamycin Complex 1 (mTORC1). The assembly of the TTT-Pontin/Reptin complex is responsive to changes in energy status. In energy sufficient conditions, the TTT-Pontin/Reptin complex promotes mTORC1 dimerization and mTORC1-Rag interaction, which are critical for mTORC1 activation. We show that WAC is a regulator of energy-mediated mTORC1 activity. In a Drosophila screen designed to isolate mutations that cause neuronal dysfunction, we identified wacky, the homolog of WAC. Loss of Wacky leads to neurodegeneration, defective mTOR activity and increased autophagy. Wacky and WAC have conserved physical interactions with mTOR and its regulators, including Pontin and Reptin which bind to the TTT complex to regulate energy-dependent activation of mTORC1. WAC promotes the interaction between TTT and Pontin/Reptin in an energy-dependent manner, thereby promoting mTORC1 activity by facilitating mTORC1 dimerization and mTORC1-Rag interaction.

Podosome formation impairs endothelial barrier function by sequestering zonula occludens proteins

“…To interfere with their dimerization ability in a dominant‐negative manner, a construct that expressed the PDZ2 domain of ZO‐1 was created (Figure S4b). A protein binding competition assay was performed as previously described (David‐Morrison et al, ). Indeed, the binding affinity between myc‐tagged ZO‐1 and FLAG‐tagged ZO‐2 was dramatically reduced when expression of the HA‐tagged PDZ2 construct was increased (Figure b,c).…”

“…To interfere with their dimerization ability in a dominantnegative manner, a construct that expressed the PDZ2 domain of ZO-1 was created ( Figure S4b). A protein binding competition assay was performed as previously described (David-Morrison et al, 2016).…”

Integration of Drosophila and Human Genetics to Understand Notch Signaling Related Diseases

“…To date, we have rescued a number of fly mutants by ubiquitous expression of human cDNAs [420–422,58,423–425], suggesting that many human genes have shared molecular functions and can replace the fly orthologs in vivo . A more rudimentary humanization experiment can be performed by co-expressing an RNAi against a fly gene together with a human cDNA.…”

The Intracranial Aneurysm Gene THSD1 Connects Endosome Dynamics to Nascent Focal Adhesion Assembly

“…Immunoprecipitates or total cell lysates (TCL) were subjected to immunoblotting. Two-step co-immunoprecipitation was described as previously reported [12,13].…”

“…To determine if THSD1 forms a multimeric protein complex with FAK-talin-vinculin rather than binding to them separately, we performed a two-step co-immunoprecipitation protocol as described previously [12]. First, we used anti-FLAG antibody to pull-down THSD1 2C).…”