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Detection of Novel Rho GAP/GTPase Selectivities Using In Vitro Split‐luciferase Assays
Author(s) -
Wilander Benjamin A.,
Hamann Michael J.
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.661.12
Subject(s) - rhoa , gtpase , cdc42 , guanine nucleotide exchange factor , microbiology and biotechnology , gtpase activating protein , dock , small gtpase , rac1 , in vitro , biology , ras superfamily , chemistry , signal transduction , biochemistry , g protein , gtp' , enzyme
Rho GTPases regulate the assembly of cellular actin structures for a wide range of dynamic activities, including actin‐dependent migration, constriction/scission and transport‐related processes. These events are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase‐activating proteins (GAPs). Although most research has tended to focus on the role of GEF‐catalyzed GTPase signal activation, GAP inactivation of GTPase signaling pathways needs to be better characterized since inactivation is required to avoid prolonged and possibly detrimental cellular responses. To this end, the GTPase selectivity of several novel hematopoietically‐expressed RhoGAPS, specifically ARHGAP1, 9, 12, 17, 25, and 26, were tested by generating MBP fusion proteins of the GAP domains of these proteins and testing their activities with Cdc42, Rac1 and RhoA. Activities were assayed using a previously described in vitro split‐luciferase assay with bacterially‐expressed purified proteins, and GAPs with known selectivities were used as positive controls. These results will provide an important first step in linking these RhoGAPs to particular GTPase pathways and eventually linking them to cellular functions. Support or Funding Information Support was provided by the Nielson Foundation, Bemidji, MN and a grant from Regenerative Medicine Minnesota (RMM‐2017‐EP‐04) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .