Role of G-proteins and S/T phosphorylation sites in the transition of Activator of G-Protein signaling 3 to cell puncta

Abstract

Activator of G-protein Signaling 3 (AGS3) exhibits broad functional diversity and oscillates among different subcellular compartments in a regulated manner. AGS3 consists of a tetratricopeptide repeat (TPR) domain and a G-protein regulatory (GPR) domain. We tested the hypothesis that phosphorylation of the AGS3 GPR domain regulated its subcellular distribution and functionality. In contrast to the cortical and/or diffuse non-homogeneous distribution of WT AGS3. AGS3 lacking 24 S/T phosphorylation sites in the GPR domain localized to cytosolic puncta and this was dependent upon a single amino acid (T602). The punctate distribution of AGS3-T602A was rescued by co-expression of Gαi/o. but not Gαs or Gαq. AGS3-T602A or WT AGS3 following treatment with alkaline phosphatase both exhibited a gel shift by SDS-PAGE as compared to untreated WT AGS3. The punctate distribution of AGS3-T602A was lost with the conversion construct AGS3-A602T*, but was still present upon T602 substitution with glutamate or aspartate. These data implicate dynamic phosphorylation as a discrete mechanism to regulate the subcellular distribution of AGS3 and associated functionality.