Investigating signaling processes in membrane trafficking.

Abstract

Signaling and phosphorylation can be very difficult areas to explore, as there can be a lot of cross-talk between signaling pathways, and the stoichiometry of phosphorylation is often very low, and is typically transient. Here we describe an innovative assay using an immunoprecipitation approach, followed by a kinase assay, coupled with a phosphorylated substrate-specific antibody. We also indicate a database and prediction program that can be used in these situations.We apply these methods to investigate the regulation of ER-to-Golgi trafficking by protein phosphorylation of critical components in the trafficking machinery. Key components of this transport step are well known thanks to the pioneering work of the 2013 Nobel Prize winners James Rothman, Randy Schekman, and Thomas Südhof. However, the regulation aspect of this process is relatively unexplored.