Palmitoylation of RGS20 Affects its Cellular Function

Abstract

Regulator of G protein signaling 20 (RGS20) is a member of the RZ family of the RGS protein superfamily, which serve as key negative regulators of G protein‐mediated signal transduction. Previous studies showed that overexpression of RGS20 is correlated with progression of triple‐negative breast cancer (TNBC) in tumor tissues and contributes to breast cancer cell migration and invasion. However, there is still a large gap relating to the regulation and function of RGS20 due to its late discovery. RGS20 contains a poly cysteine string motif, which is assumed to be heavily palmitoylated. Palmitoylation, an important post‐translational protein modification, plays an important role in cells by changing cellular functions of proteins. Consequently, in this study we determined palmitoylation regulation of RGS20, which could provide more information for new potential treatment strategies for TNBC. We used click chemistry to show that RGS20 is palmitoylated in transfected HEK‐293T cells and MDA‐MB‐231 triple negative breast cancer cells. We also showed that constitutively activated Gαo, GαoQ205L, significantly increases palmitoylation levels of RGS20. Furthermore, palmitoylated RGS20 associates much more with GαoQ205L, while de‐palmitoylated RGS20 lost the association with GαoQ205L. Next, we detected how palmitoylation affects RGS20 GAP activity, as well as cAMP signaling downstream of Gαo. We mapped the palmitoylation site crucial for all of these effects to a conserved cysteine in the RGS domain. Mutating this cysteine residue resulted in a significant decrease in both palmitoylation signal and the association with GαoQ205L. Overall, we showed that palmitoylation affects functions of RGS20 in cells and that a conserved cysteine in the RGS domain is a critical site for its palmitoylation.