Abstract

The G12/13 subfamily of heterotrimeric guanine nucleotide binding proteins is composed of Gα12 and Gα13, which play important intracellular signaling roles, including activation of serum response factor (SRF). SRF activates transcription of serum response element (SRE) regulated genes involved in cell growth and tumorigenic events. Previous studies have reported serine phosphorylation occurs near the N-terminus of Gα12, and a specific threonine within the Switch I region of Gα13 serves as a phosphorylation site for Protein Kinase A (PKA). To identify and investigate the roles of these residues on SRE signaling, point mutations were engineered in Gα12 and Gα13 to either abolish or mimic phosphorylation. Replacing a Gα12-specific N-terminal serine with the phosphomimic aspartic acid disrupted Gα12 signaling to SRE, as measured by firefly luciferase assays. Signaling by Gα13 was unaffected by phosphomimics of potentially homologous serines, suggesting a Gα12-specific mechanism. Insertion of the Gα12 N- terminus harboring the phosphomimic into Gα13 bestowed negative regulation of SRE signaling upon this chimeric protein. These results point to a kinase-dependent switching mechanism at the N-terminus of Gα12 that governs its growth signaling. The ability of the phosphomimic-containing Gα12 to activate SRF was rescued by inserting a sequence allowing covalent attachment of a myristoyl lipid, suggesting the phosphomimic interferes with palmitoylation of the G protein. To investigate the effects of PKA on SRE signaling, the catalytic subunit of PKA was overexpressed in tandem with phosphomimics and blocks within the Switch I region of Gα12 and Gα13. PKA dramatically increased growth signaling by both G proteins, but this effect was mitigated in the presence of a Thr-to- Ala block suggesting that phosphorylation by PKA within the Switch I region of G12/13 proteins stimulates basal cell growth. Together, these kinase dependent switches may regulate G12/13 activity and serve as targets for anti-cancer therapies.

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