Distribution of Activator of G‐Protein Signaling 3 within the Aggresomal Pathway: Role of Serine/Threonine Residues in the G‐Protein Regulatory Domain and Lysosomal Processing
Authors: Vural Ali , Oner S. Sadik , Ma Dzwokai , Lanier Stephen M. 
Published: November 2023

Activator of G‐protein Signaling 3 (AGS3), a receptor independent activator of G‐protein signaling, consists of 7 tetratricopeptide repeats (TPR) and 4 G‐protein regulatory motifs (GPR) separated by a linker region. Disruption of TPR organization by point and deletion mutations stabilizes the protein in the aggresomal pathway. As an extension of this work and as part of a broader strategy to define regulatory factors affecting AGS3's subcellular “positioning”, we next examined a potential role for AGS3 phosphorylation with an initial focus on the GPR domain. AGS3 is a phosphoprotein, but sites of phosphorylation are not defined. We previously examined the subcellular location of AGS3 construct in which the 24 S/T residues in the GPR domain of the protein were mutated to alanine [phosphomutant (PM)1] and transiently expressed in COS‐7 and HEK‐293 cells. In contrast to the distribution of WT‐AGS3‐GFP to the cell cortex accompanied by a diffuse distribution in the cytosol, AGS3‐GFP‐PM1 distributed to pre‐aggresomal punctate structures in the cytoplasm and SDS‐PAGE and immunoblots of cell lysates indicated a migration shift for AGS3‐GFP‐PM1 versus AGS3‐GFP. Following cell treatment with the proteasome inhibitor MG132, AGS3‐PM1 localized to a perinuclear aggresome. To identify specific S/T residues in the GPR domain that regulate this subcellular distribution, we generated a subset of AGS3‐GFP‐PM constructs and examined their subcellular distribution following transient expression in COS‐7 and HEK‐293. Mutation of a single residue (T602A), which is located between GPR‐III and GPR‐IV was sufficient to localize AGS3 to the pre‐aggresomal punctate structures. The subcellular distribution of AGS3‐GFP‐T602A or AGS3‐GFP‐PM1 was not altered by the G[beta‐gamma] antagonist gallein (10 mM) or cell pretreatment with pertussis toxin (200ng/ml), but coexpression of G[alpha] prevented the observed distribution of the two constructs to the pre‐aggresomal punctate structures. The subcellular distribution of WT‐AGS3 was not altered by PI3K inhibitors (3‐MA (5 mM, 24 hrs), wortmannin (50 nM, 24 hrs), LY‐294,002 (10 mM, 24 hrs)) and mTOR inhibitor (rapamycin (100 nM, 24 hrs)) but inhibition of lysosome acidification by cell treatment with ammonium chloride (25 mM, 24 hrs) localized WT‐AGS3 to the pre‐aggresomal punctate structures. These data indicate that a single residue mutation in the GPR domain or in the TPR domain of AGS3 or lysosome inhibition either promote entry of AGS3 into the aggresomal pathway and/or stabilize the protein in pre‐aggresomal punctate structures. The appearance of AGS3 mutants in the pre‐aggresomal punctate structures is not observed upon interaction of the protein with its binding partner G[alpha], which together with the data generated with TPR and GPR single residue mutations, indicate that the movement of the protein into or within the aggresomal pathway may be modulated by specific signaling pathways.