This proposal focuses on an emerging signal transduction paradigm which counters the prevailing view that the nucleus is a passive recipient of signals produced at the plasma membrane. Our laboratory and others have shown that receptor activation at the cell surface results in regulation of nuclear enzymes that produce and modify potent lipid signaling molecules. While this area has garnered increasing interest, we still understand neither the molecular aspects of the regulation nor the precise physiological roles of the induced lipid metabolism. The proposed studies aim at elucidating key elements of this signal transduction mechanism and its functional consequences. We find that the small molecular weight GTP-binding protein RhoA reciprocally regulates two signaling enzymes, diacylglycerol kinase-theta and phospholipase D, at the nucleus in response to alpha-thrombin stimulation of fibroblasts. We have termed this regulation the """"""""RhoA Switch"""""""". We predict that the resulting modulation of diacylglycerol and phosphatidic acid levels and molecular composition have important physiological consequences such as the regulation of nuclear localization and activity of protein kinase C-alpha. Two heterotrimeric GTP-binding proteins, Gq and G13, and the lipid kinase PI 3-kinase mediate thrombin activation of this pathway. We hypothesize that; alpha-thrombin, after initiating an early Gq-dependent increase in nuclear diacylglycerol kinase-theta activity, stimulates a G13/PI 3-kinase-dependent nuclear translocation of RhoA that acts as a temporal """"""""switch"""""""" to inhibit nuclear diacylglycerol kinase-theta while activating nuclear phospholipase D. We will test the hypothesis that Gq is essential for the induced increase in nuclear diacylglycerol kinase-theta activity, and identify the diacylglycerol-theta domains essential for its nuclear translocation in Aim I.
Aim II will determine the molecular mechanism of the RhoA Switch by testing whether PI 3-kinase activation is responsible for nuclear RhoA translocation. Further, we will determine the molecular interactions between RhoA, diacylglycerol kinase-theta and phospholipase D. The physiological consequences of the RhoA Switch will be examined in Aim III. We will focus on the effect of the Switch on nuclear protein kinase C-alpha activity, composition of nuclear phosphatidic acids, and growth. We believe that an understanding of nuclear lipid signaling is key to our ability to intervene in events such as wound repair and tissue remodeling.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM059251-08S1
Application #
7684399
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Chin, Jean
Project Start
1999-05-01
Project End
2010-07-31
Budget Start
2008-08-01
Budget End
2010-07-31
Support Year
8
Fiscal Year
2008
Total Cost
$12,841
Indirect Cost
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Petro, Elizabeth J; Raben, Daniel M (2013) Bacterial expression strategies for several Sus scrofa diacylglycerol kinase alpha constructs: solubility challenges. Sci Rep 3:1609
Tu-Sekine, Becky; Goldschmidt, Hana; Petro, Elizabeth et al. (2013) Diacylglycerol kinase ?: regulation and stability. Adv Biol Regul 53:118-26
Tu-Sekine, Becky; Raben, Daniel M (2012) Dual regulation of diacylglycerol kinase (DGK)-?: polybasic proteins promote activation by phospholipids and increase substrate affinity. J Biol Chem 287:41619-27
Link, Tiffany M; Park, Una; Vonakis, Becky M et al. (2010) TRPV2 has a pivotal role in macrophage particle binding and phagocytosis. Nat Immunol 11:232-9
Tu-Sekine, Becky; Raben, Daniel M (2010) Characterization of cellular DGK-theta. Adv Enzyme Regul 50:81-94
Raben, Daniel M; Wattenberg, Binks W (2009) Signaling at the membrane interface by the DGK/SK enzyme family. J Lipid Res 50 Suppl:S35-9
Raben, Daniel M; Tu-Sekine, Becky (2008) Nuclear diacylglycerol kinases: regulation and roles. Front Biosci 13:590-7
Tu-Sekine, Becky; Ostroski, Michele; Raben, Daniel M (2007) Modulation of diacylglycerol kinase theta activity by alpha-thrombin and phospholipids. Biochemistry 46:924-32
Tu-Sekine, Becky; Ostroski, Michele; Raben, Daniel M (2006) Analysis of two diacylglycerol kinase activities in mixed micelles. Adv Enzyme Regul 46:12-24
Wattenberg, Binks W; Pitson, Stuart M; Raben, Daniel M (2006) The sphingosine and diacylglycerol kinase superfamily of signaling kinases: localization as a key to signaling function. J Lipid Res 47:1128-39

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