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Published online 19 March 2001. doi:10.1083/jcb.152.6.1135
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© The Rockefeller University Press, 0021-9525/2001//1135 $5.00
The Journal of Cell Biology, Volume 152, Number 6, , 2001 1135-1144

Original Article

Diacylglycerol Kinase {zeta} Regulates Ras Activation by a Novel Mechanism



Matthew K. Tophama and Stephen M. Prescotta

a The Huntsman Cancer Institute and Department of Internal Medicine, University of Utah, Salt Lake City, Utah 84112
The Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84132.(801) 585-6345(801) 585-3401

stephen.prescott{at}hci.utah.edu

Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGK{zeta}, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK{zeta} also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK{zeta} and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGK{zeta} in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGK{zeta}, which controls local accumulation of DAG that would otherwise activate RasGRP.

Key Words: diacylglycerols • diacylglycerol kinase • signal transduction • H-Ras oncogenes • RasGRP protein

© 2001 The Rockefeller University Press

Abbreviations used in this paper: CMV, cytomegalovirus; DGK, DAG kinase; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GRP, guanyl nucleotide-releasing protein (GRP); HA, hemagglutinin; PA, phosphatidic acid; PMA, phorbol 12-myristate 13-acetate; TCR, T cell receptor.


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