Overexpressed Gα13 activates serum response factor through stoichiometric imbalance with Gβγ and mislocalization to the cytoplasm

• Published in: Cellular signalling Vol. 102; p. 110534
• Main Authors: Hasan, Sharmin, White, Nicholas F., Tagliatela, Alicia C., Durall, R. Taylor, Brown, Katherine M., McDiarmid, Gray R., Meigs, Thomas E.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.02.2023
• Subjects: Cytoplasm - metabolism; GNA13; GTP-Binding Protein alpha Subunits, G12-G13 - metabolism; GTP-Binding Proteins - metabolism; Gα13; HEK293 Cells; Heterotrimeric G protein; Humans; Neoplasms; Palmitoylation; Serum response element; Serum response factor; Serum Response Factor - metabolism; PBS; Gα13; GTP; Palmitoylation; SRF; SRE; MRTFA; Serum response element; E-cadherin; HEK; TIC; GNA13; GFP; Hsp90; Heterotrimeric G protein; DMEM; SDS-PAGE; GDP; RhoGEF; RH; Serum response factor; GPCR; GAPDH; G protein
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2022.110534

Gα13, a heterotrimeric G protein α subunit of the G12/13 subfamily, is an oncogenic driver in multiple cancer types. Unlike other G protein subfamilies that contribute to cancer progression via amino acid substitutions that abolish their deactivating, intrinsic GTPase activity, Gα13 rarely harbors such mutations in tumors and instead appears to stimulate aberrant cell growth via overexpression as a wildtype form. It is not known why this effect is exclusive to the G12/13 subfamily, nor has a mechanism been elucidated for overexpressed Gα13 promoting tumor progression. Using a reporter gene assay for serum response factor (SRF)-mediated transcription in HEK293 cells, we found that transiently expressed, wildtype Gα13 generates a robust SRF signal, approximately half the amplitude observed for GTPase-defective Gα13. When epitope-tagged, wildtype Gα13 was titrated upward in cells, a sharp increase in SRF stimulation was observed coincident with a “spillover” of Gα13 from membrane-associated to a soluble fraction. Overexpressing G protein β and γ subunits caused both a decrease in this signal and a shift of wildtype Gα13 back to the membranous fraction, suggesting that stoichiometric imbalance in the αβγ heterotrimer results in aberrant subcellular localization and signalling by overexpressed Gα13. We also examined the acylation requirements of wildtype Gα13 for signalling to SRF. Similar to GTPase-defective Gα13, S-palmitoylation of the wildtype α subunit was necessary for SRF activation but could be replaced functionally by an engineered site for N-terminal myristoylation. However, a key difference was observed between wildtype and GTPase-defective Gα13: whereas the latter protein lacking palmitoylation sites was rescued in its SRF signalling by either an engineered polybasic sequence or a C-terminal isoprenylation site, these motifs failed to restore signalling by wildtype, non-palmitoylated Gα13. These findings illuminate several components of the mechanism in which overexpressed, wildtype Gα13 contributes to growth and tumorigenic signalling, and reveal greater stringency in its requirements for post-translational modification in comparison to GTPase-defective Gα13. [Display omitted] •Gα13 drives signalling to Serum Response Factor (SRF) as an overexpressed, wildtype form.•Overexpressed Gα13 “spills over” from a membrane-bound to a cytoplasmic location.•Transfection with beta and gamma subunits suppresses aberrant signalling by wildtype Gα13.•Wildtype and mutationally-active Gα13 have distinct lipidation requirements for SRF signalling.