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MBC in Press, published online ahead of print December 27, 2007
Mol. Biol. Cell 10.1091/mbc.E06-08-0737

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Submitted on August 22, 2006
Revised on November 15, 2007
Accepted on December 14, 2007

StARD13(Dlc-2) RhoGap Mediates Ceramide Activation of Phosphatidylglycerolphosphate Synthase and Drug Response in Chinese Hamster Ovary Cells

Grant M. Hatch,*{dagger} Yuan Gu,{ddagger} Fred Y. Xu,* Jeannick Cizeau,{ddagger} Shannon Neumann,{ddagger} Ji-Seon Park,{ddagger} Shauna Loewen,{ddagger}{dagger} and Michael R.A. Mowat{ddagger}{dagger}

{ddagger}Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, MB, Canada, R3E 0V9; Departments of {dagger}Biochemistry and Medical Genetics and *Pharmacology and Therapeutics, University of Manitoba, Winnipeg, MB, Canada, R3E 0V9

Monitoring Editor: Howard Riezman

To identify genes involved in etoposide drug response, we used promoter trap mutagenesis to isolate an etoposide resistant CHO cell line. This resistant CHO-K1 line, named E91, showed cross resistance to C2-ceramide (N-acetylsphingosine). The promoter trap retrovirus was found integrated into intron one-two of the Dlc-2 (Stard13) RhoGap gene. The 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 A2 enzyme activities in cells after C2-ceramide addition. Parental cells showed elevated enzyme activities after treatment with C2-ceramide or tumor necrosis factor % but, not the E91 cells. These results suggested 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. The RNAi knock down cell line also showed increased etoposide resistance. This study is the first report for the regulation of a phospholipid biosynthetic enzyme through RhoGap expression.

Address correspondence to: Michael R.A. Mowat (mmowat{at}cc.umanitoba.ca)






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