Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, USA.
J Cell Sci. 2018 Dec 5;131(23):jcs216507. doi: 10.1242/jcs.216507.
Activator of G-protein signaling 3 (AGS3, also known as GPSM1) 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. Here, we tested the hypothesis that phosphorylation of the AGS3 GPR domain regulates its subcellular distribution and functionality. In contrast to the cortical and/or diffuse non-homogeneous distribution of wild-type (WT) AGS3, an AGS3 construct lacking all 24 potential phosphorylation sites in the GPR domain localized to cytosolic puncta. This change in localization was revealed to be dependent upon phosphorylation of a single threonine amino acid (T602). The punctate distribution of AGS3-T602A was rescued by co-expression of Gα and Gα but not Gα or Gα Following treatment with alkaline phosphatase, both AGS3-T602A and WT AGS3 exhibited a gel shift in SDS-PAGE as compared to untreated WT AGS3, consistent with a loss of protein phosphorylation. The punctate distribution of AGS3-T602A was lost in an AGS3-A602T conversion mutant, but was still present upon T602 mutation to glutamate or aspartate. These results implicate dynamic phosphorylation as a discrete mechanism to regulate the subcellular distribution of AGS3 and associated functionality.
G 蛋白信号转导激活因子 3（AGS3，也称为 GPSM1）表现出广泛的功能多样性，并以受调控的方式在不同的亚细胞隔室之间振荡。AGS3 由四肽重复（TPR）结构域和 G 蛋白调节（GPR）结构域组成。在这里，我们检验了 AGS3 GPR 结构域磷酸化调节其亚细胞分布和功能的假说。与野生型（WT）AGS3 的皮质和/或弥散非均匀分布相反，缺乏 GPR 结构域中所有 24 个潜在磷酸化位点的 AGS3 构建体定位于细胞质点状。这种定位变化被证明依赖于单个苏氨酸氨基酸（T602）的磷酸化。AGS3-T602A 的点状分布可通过共表达 Gα 和 Gα 得到挽救，但不能通过共表达 Gα 或 Gα 得到挽救。在用碱性磷酸酶处理后，与未经处理的 WT AGS3 相比，AGS3-T602A 和 WT AGS3 在 SDS-PAGE 中均表现出凝胶迁移，这与蛋白质磷酸化的丧失一致。AGS3-T602A 的点状分布在 AGS3-A602T 转换突变体中丢失，但在 T602 突变为谷氨酸或天冬氨酸时仍存在。这些结果表明动态磷酸化是调节 AGS3 及其相关功能的亚细胞分布的离散机制。
