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Overexpressed Gα13 stimulates Serum Response Factor mediated transcription concurrent with relocalization to a soluble fraction
Author(s) -
McDiarmid Gray R,
Tagliatela Alicia C,
Meigs Thomas E.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.865.3
Subject(s) - wild type , heterotrimeric g protein , serum response factor , transcription factor , hek 293 cells , signal transduction , protein subunit , microbiology and biotechnology , chemistry , biology , g protein , mutant , receptor , biochemistry , gene
Heterotrimeric G proteins consist of α, β, and γ subunits, and activating, GTPase‐deficient mutations in the α subunit of several classes have been implicated in tumor progression. The G12/13 class of α subunits, comprising Gα12 and Gα13, has the unique ability to stimulate oncogenic transformation of cultured cells though overexpression of the wildtype α subunit. However, the mechanism that allows this signaling property of wildtype Gα12 and Gα13 is not understood. Using transiently transfected HEK293 cells, we examined varying levels of constitutively active and wildtype forms of Gα13 for ability to stimulate serum response factor (SRF) mediated transcription, a cell growth signaling pathway implicated in melanoma and other cancers. Constitutively active Gα13 showed an approximate 100‐fold stimulation of SRF signaling, and unexpectedly, we observed a response fully half this magnitude using wildtype Gα13. In cell fractionation experiments using an epitope‐tagged wildtype Gα13, this transiently expressed protein began appearing in the soluble fraction as its levels increased, whereas endogenous Gα13 remained in the membrane‐associated fraction. Under conditions in which Gα13 redistribution was observed, SRF activation showed a sharp increase. This stimulatory effect of wildtype Gα13 was blunted by co‐expression of Gβ 1 and Gγ 2 subunits, suggesting overexpressed Gα13 drives SRF signaling due to stoichiometric imbalance in the heterotrimer. Conversely, SRF stimulation by wildtype Gα12 was enhanced by these Gβ/Gγ subunits. Gβ 1 /Gγ 2 co‐expression also changed the subcellular distribution of wildtype Gα13 to a predominantly membrane‐associated state. Furthermore, mutation of two N‐terminal Cys residues required for Gα13 palmitoylation caused localization to the soluble fraction but abolished SRF signaling, suggesting this lipid modification as critical for the soluble, overexpressed pool of Gα13 to drive this growth pathway. Support or Funding Information We acknowledge support from the GlaxoSmithKline and C.D. Spangler Foundations, Lineberger Comprehensive Cancer Center, and UNC‐Asheville Undergraduate Research Program.