Visual screening and analysis for kinase-regulated membrane trafficking pathways that are involved in extensive β-amyloid secretion

Membrane trafficking is an important cellular process that enables the precise localization of membrane proteins. The disturbance of membrane trafficking results in various disease states. To explore systematically the defects in trafficking pathways that cause these disturbances or disease states, we developed an automated high-throughput fluorescence-based imaging system and carried out visual screening for kinase-regulated trafficking pathways of the cation-independent mannose 6-phosphate receptor (CI-M6PR) in HeLa cells. As the result of our visual screening, which examined the effect of kinase inhibitors and a kinase siRNA library, we identified five kinases (CDC42BPB, PRKACA, PRKACG, GSK3 beta and CSNK2A1) that regulate CI-M6PR trafficking. Moreover, we focused on Alzheimer's disease (AD) to study the relationship between the five kinases and a disease state. Notably, two trafficking pathways, which were regulated by PRKACG and GSK3 beta, respectively, induced high levels of secretion of A beta, the hallmark of AD. In addition, we found that the modulation of GSK3 beta activity affected the microtubule plus end tracking function of cytoplasmic linker protein-associating protein 2 and resulted in the perturbation of BACE1 localization/trafficking and extensive A beta secretion. Our systems provide new approaches for the analysis of spatially-regulated membrane trafficking and related disease states.