Abstract

Heterotrimeric guanine nucleotide binding proteins (G proteins) are signal transducers that have been found to have mechanistic roles either as downstream conduits of integral membrane protein receptors or regulators of various intracellular processes. In particular, the G12/13 subfamily has been implicated in a range of specialized cell growth signaling pathways. The members of this subfamily, Gα12 and Gα13, utilize novel mechanisms with regard to serine/threonine mediated phosphorylation, and recent work in our laboratory has revealed new aspects of this regulation within the G12/13 subfamily. However, regulation of these proteins by tyrosine phosphorylation has not been reported. A recent proteomic study by Chakravorty et al. (2018) identified a likely tyrosine phosphorylation event at position 44 in Gα13. To investigate this possible regulatory mechanism within Gα13, a point mutation was engineered to replace tyrosine at the 44th position with a phenylalanine, thus eliminating the hydroxyl group that allows phosphorylation with minimal disruption to the structural integrity of Gα13. At present, we are confirming this mutant through DNA sequencing. Future work with the successfully constructed Y44F mutant will focus on detecting signaling changes that occur due to this mutation. These experiments will include transcriptional activation assays to measure Gα13 signaling effects on cell growth, as well as protein interaction experiments to gauge any disruption of Gα13 binding to other proteins. Additionally, Gα12 and invertebrate G12/13 homologs will be analyzed in parallel to find if these proteins share a tyrosine-dependent regulatory mechanism with Gα13.

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