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Effects of Differential Acylation on Aberrant Growth Signaling by Overexpressed Gα13
Author(s) -
White Nicholas F.,
McDiarmid Gray R.,
Tagliatela Alicia C.,
Meigs Thomas E.
Publication year - 2018
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.2018.32.1_supplement.661.3
Subject(s) - palmitoylation , myristoylation , wild type , heterotrimeric g protein , signal transduction , hek 293 cells , g protein , chemistry , microbiology and biotechnology , biology , cysteine , biochemistry , receptor , phosphorylation , gene , mutant , enzyme
Overexpression of Gα13, a member of the G12/13 subfamily of heterotrimeric G proteins, has been implicated in cell transformation and the progression of several cancer types. The mechanism through which wildtype, overexpressed Gα13 drives aberrant growth signaling is not known. G protein α subunits are modified post‐translationally by acylation at the N‐terminus, and this addition of a palmitoyl and/or myristoyl group occurs for all mammalian Gα proteins. Gα13 was previously engineered to mutate two cysteine residues necessary for dual palmitoylation. This modification abrogated signaling to serum response factor by Gα13 in both its wildtype and constitutively activated forms. To determine whether myristoylation restores signaling to non‐acylated wildtype Gα13, the protein was modified to harbor the 6 N‐terminal amino acids from GαT, which is myristoylated but not palmitoylated. Loss of growth signaling by non‐palmitoylated, wildtype G13 was rescued by introduction of this myristoylated sequence. In addition, we used cellular fractionation to assess intracellular distribution of these Gα13 variants. Overexpressed, wildtype Gα13 showed a shift from a membrane associated fraction to a soluble pool, which correlated with a sharp increase in serum response factor signaling. Gα13 lacking any acylation sites localized entirely to the soluble fraction but exhibited no growth signaling. We have also examined the role of an N‐terminal polybasic motif in overexpressed wildtype Gα13 signaling, through amino acid substitutions in this region. To query the nucleotide bound state of the soluble Gα13 pool, we combined cell fractionation with trypsin digestion assays. Surprisingly, overexpressed wildtype Gα13 in the soluble fraction was fully degraded, suggesting lack of GTP binding. These constructs have facilitated co‐precipitation experiments to determine whether change in acylation state affects Gα13 binding to specific target proteins. Support or Funding Information We acknowledge support from the North Carolina GlaxoSmithKline Foundation, the C.D. Spangler Foundation, UNC Lineberger Comprehensive Cancer Center, and the UNC‐Asheville Undergraduate Research Program. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .