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RhoGAP mediates ceramide activation of phosphatidylglycerolphosphate synthase and drug response in Chinese hamster ovary cells
Author(s) -
Hatch Grant Michael,
Gu Yuan,
Xu Fred Y,
Cizeau Jeannick,
Neumann Shan,
Park JiSeon,
Loewen Shauna,
Mowat Michael R. A.
Publication year - 2008
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.22.2_supplement.269
Subject(s) - rhoa , ceramide , chinese hamster ovary cell , ceramide synthase , biology , lipid signaling , microbiology and biotechnology , cell culture , transfection , signal transduction , biochemistry , enzyme , apoptosis , genetics
Promoter trap mutagenesis was used to isolate an etoposide resistant CHO cell line. This resistant CHO‐K1 line, named E91, showed cross resistance to C2‐ceramide (N‐acetylsphingosine) mediated cell killing. The promoter trap retrovirus integrated into intron one‐two of the Dlc‐2 ( Stard13 ) RhoGap gene. E91 cells showed elevated GTP bound RhoA levels compared with the parental line suggesting that retrovirus integration had inactivated one of the Dlc‐2 RhoGap alleles. To test if E91 cells were impaired in an intracellular ceramide‐regulated process not directly related to cell killing, we measured mitochondrial phosphatidylglycerolphosphate (PGP) synthase and phospholipase A 2 enzyme activities in cells after C2‐ceramide addition. Parental cells showed elevated enzyme activities after treatment with C2‐ceramide or TNFα but, not the E91 cells. These results indicate that intracellular ceramide signaling was defective in E91 cells due to increased levels of active GTP bound RhoA. RNA knockdown experiments of the Dlc2 RhoGap resulted in increased GTP bound RhoA and reduced induction of PGP synthase after C2‐ceramide addition compared with controls. Expression of a dominant negative RhoA in the E91 cell line allowed induction of PGP synthase by ceramide. This study is the first report for the regulation of a phospholipid biosynthetic enzyme through a G protein. (Supported by the CIHR, NCIC, G.M.H. is a Canada Research Chair in Molecular Cardiolipin Metabolism).