A fluorescent protein-readout for transcriptional activity reveals regulation of APP nuclear signaling by phosphorylation sites.

A fluorescent protein-readout for transcriptional activity reveals regulation of APP nuclear signaling by phosphorylation sites. Biol Chem. 2019 May 01;: Authors: Konietzko U, Gersbacher MT, Streuli J, Krüger M, Thöni S, Kins S, Nitsch RM Abstract Signaling pathways that originate at the plasma membrane, including regulated intramembrane proteolysis (RIP), enable extracellular cues to control transcription. We modified the yeast Gal4 transcription system to study nuclear translocation of transcriptionally active complexes by using the fluorescent protein Citrine (Cit) as a reporter. This enabled highly sensitive quantitative analysis of transcription in situ at the single cell level. The Gal4/UAS-Cit transcription assay displayed a sigmoidal response limited by the number of integrated reporter cassettes. We validated the assay by analyzing nuclear translocation of the amyloid precursor protein (APP) intracellular domain (AICD) and confirmed the requirement of Fe65 for nuclear translocation of AICD. In addition to strong on-off effects on transcriptional activity, the results of this assay establish that phosphorylation modifies nuclear signaling. The Y682F mutation in APP showed the strongest increase in Cit expression, underscoring its role in regulating Fe65 binding. Together, we established a highly sensitive fluorescent protein-based assay allowing to monitor transcriptional activity at the single cell level and demonstrating ...
Source: Biological Chemistry - Category: Chemistry Tags: Biol Chem Source Type: research