Abstract

Heterotrimeric G proteins of the Gɑ12/13 subfamily regulate cell proliferation, and have been implicated in cancer development. While mutations of G proteins are often implicated as drivers of tumorigenesis, simple overexpression of Gɑ13 causes cancerous signaling, and has been shown to aid cancer cells in developing tumor-initiating cell (TIC) properties. In order for signaling and membrane localization to occur for most G protein ɑ subunits, enzymatic attachment of the fatty acid palmitate to one or more cysteine side chains near the N-terminus is required. For Gɑ13, cysteines at positions 14 and 18 in this 377-amino acid protein have been shown to undergo this palmitoylation step, however, the roles of the individual cysteine modifications in Gɑ13 signaling function have not been reported. In order for the specific requirements for membrane localization and signaling of Gɑ13 to be investigated, cysteine-to-alanine mutations were engineered in Gɑ13 at the individual positions (14 and 18) and in a mutant combining both mutations. Introduction of negative charges around these key cysteine residues was also investigated through mutations of surrounding uncharged amino acids to aspartic acid residues. These Gɑ13 variants were confirmed by DNA sequencing and transfected into human embryonic kidney (HEK) cells. Ability of Cys mutants to participate in cancerous growth signaling was assessed through serum response factor (SRF) measurements, a well-established readout of oncogenic growth signaling in human cells. Effects of Cys to Ala mutations and introduction of negative charges on the intracellular localization of Gɑ13 were also determined, using cell fractionation techniques. Results suggest that mutation of either cysteine residue completely disables SRE signaling, yet does not preclude Gɑ13 from membrane localization. The differences in membranous vs. cytoplasmic distribution for these mutants are interesting, as they suggest individual Cys residues play roles in tumorigenic signaling beyond simply regulating Gɑ13 association with the cell membrane.

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