A phosphorylation mimic at the Gα12 N-terminus inhibits palmitoylation and signaling to serum response factor

Bailey Cook

Biology

Thomas E. Meigs

The G12/13 subfamily of heterotrimeric guanine nucleotide binding proteins (G proteins), is composed of Gα12 and Gα13, which play important intracellular signaling roles including activation of serum response factor (SRF),. SRF binds the serum response element (SRE), of the c-fos promoter and activates genes involved in cell growth and tumorigenic events. The G12/13 N-terminal regions have not been characterized structurally; however, previous reports indicate phosphorylation of a serine in this region of Gα12. Replacing serine-9 of a GTPase-deficient Gα12 with acidic phosphorylation mimics abolished its signaling to SRF as measured by firefly luciferase assays for SRE-mediated transcription, whereas replacement with positive charge (arginine), enhanced this response. These substitutions also inhibited SRF activation by a chimera composed of the Gα12 N-terminus attached to GTPase-deficient Gα13. In addition, we introduced negatively-charged residues adjacent to the S-palmitoylation site of Gα12 (cysteine-11), required for SRF activation; these substitutions disrupted this response. These results led us to hypothesize that mutations introducing negative charge were inhibiting palmitoylation of Gα12 at cysteine-11. To test this, we engineered N-terminal myristoylation or C-terminal isoprenylation sites in the Gα12 phosphorylation mimic mutants; both of these motifs fully rescued SRF signaling. Contrary to Gα12, signaling by Gα13 was unaffected by negatively-charged amino acids engineered adjacent to its two N-terminal sites of palmitoylation, suggesting these α subunits in the same G protein subfamily undergo distinct mechanisms of palmitate attachment. We also examined the subcellular localization of the Gα12 and Gα13 phosphorylation mimic constructs. Fractionation of cells into soluble and membranous components showed a different distribution of a Gα12/Gα13 chimera in comparison to Gα13, although both proteins exhibited robust SRF signaling. The difference in location was unaffected by phosphorylation mimics at serine-9 of the chimera. Currently, we are investigating whether engineered negative charge at the N-terminus affects Gα12 interaction with specific target proteins, and also testing kinases for the ability to phosphorylate Gα12.

Jan-9-2024