Factors regulating the subcellular localization of Activators of G‐protein Signaling 3: The role of serine/threonine residues in the G‐protein regulatory domain

Abstract

Activators of G‐protein Signaling (AGS) proteins contain one to four G‐protein regulatory motifs (GPR) that serve as docking sites for Gαi/o/t. The GPR‐Gα signaling module is regulated by guanine nucleotide exchange factors and provides unexpected signaling diversity for the “G‐switch”. AGS3, which contains seven tetratricopeptide repeats (TPR) and four GPR motifs, is involved in a wide range of biological functions including asymmetric cell division, autophagy and addictive behavior. Interaction of AGS3 with Gαi or FRMPD1 stabilizes AGS3 at the cell cortex, whereas disruption of the TPR motif stabilizes the protein in the aggresome pathway. We asked if AGS3 trafficking was regulated by phosphorylation. As a first approach to this issue, we determined the subcellular distribution of a mutant GFP‐tagged AGS3 (AGS3‐GFP‐PM1) in which 24 S/T residues in the GPR domain of the protein were changed to alanine to eliminate S/T phosphorylation in this region. GFP tagged AGS3 WT, PM1 and TPR mutants were expressed in HEK‐293 cells and the subcellular distribution determined by immunofluorescence microscopy. In contrast to wild type AGS3, both the PM1 and TPR mutants distributed to preaggresomal structures in the cytosol. These data suggest that phosphorylation of AGS3 may play a regulatory role in its trafficking within the cell. Dysregulation of the subcellular location of AGS3 may contribute to pathologies involving altered protein processing through the aggresomal and autophagic pathways.