The leukemia-associated Rho guanine nucleotide exchange factor (LARG) was recently identified as a fusion partner of the mixed lineage leukemia (MLL) protein in acute myeloid leukemia. LARG is a novel member of the rapidly expanding Dbl family of oncoproteins that promote malignant transformation by activating Ras-related Rho family GTPases. Like other Dbl family proteins, LARG contains a Dbl homology (DH) domain that functions as a guanine nucleotide exchange factor and activator of Rho GTPases. The DH domain is followed by a pleckstrin homology (PH) domain that presumably regulates DH domain function. LARG also contains a regulator of G-protein signaling (RGS) domain, suggesting that it may link G protein-coupled receptor signaling to Rho GTPases. Our preliminary studies determined that LARG is an activator of RhoA and can cause transformation of NIH 3T3 mouse fibroblasts. We have proposed four specific aims to perform detailed structure-function analyses of LARG.
Specific aim 1 will determine the roles of the DH and PH domains in mediating LARG activation of RhoA. In particular, whether the PH domain regulates DH domain function in a phosphatidylinositol 3-kinase dependent fashion will be determined.
Specific aim 2 will evaluate the role of the RGS domain in linking LARG with G protein coupled receptor signaling. This includes a determination of which heterotrimeric G alpha subunit(s) is regulated by the RGS domain and which G alpha subunit(s) in turn regulates LARG DH domain activation.
Specific aim 3 will determine if the tumor-associated MLL-LARG fusion protein is an aberrantly activated form of LARG and can promote growth transformation of epithelial cells and lL-3 independent growth of 32D myeloid cells. Finally, Specific Aim 4 will involve a determination of the crystal structure of the DH/PH domains of LARG complexed with its GTPase target, RhoA, and the determination of the structural basis for DH domain recognition of GTPases. Although the number of Dbl family oncoproteins continue to increase at a rapid pace, to date, LARG is the only functional Dbl protein found to be mutated in human cancer. Our studies will provide a comprehensive, structural, biochemical, and biological analysis of LARG function and assess a role for aberrant LARG activation of RhoA in AML development.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA092240-03
Application #
6616783
Study Section
Pathology B Study Section (PTHB)
Program Officer
Mufson, R Allan
Project Start
2001-08-01
Project End
2006-07-31
Budget Start
2003-08-06
Budget End
2004-07-31
Support Year
3
Fiscal Year
2003
Total Cost
$240,450
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Krishnamurthy, Nirmala; Liu, Lili; Xiong, Xiahui et al. (2015) Downregulation of hPMC2 imparts chemotherapeutic sensitivity to alkylating agents in breast cancer cells. Cancer Biol Ther 16:518-27
Montano, Monica M; Krishnamurthy, Nirmala; Sripathy, Smitha (2012) TARGETING THE GENOTOXIC EFFECTS OF ESTROGENS. Drug Discov Today Dis Mech 9:e29-e33
Mitin, Natalia; Roberts, Patrick J; Chenette, Emily J et al. (2012) Posttranslational lipid modification of Rho family small GTPases. Methods Mol Biol 827:87-95
Krishnamurthy, N; Ngam, C R; Berdis, A J et al. (2011) The exonuclease activity of hPMC2 is required for transcriptional regulation of the QR gene and repair of estrogen-induced abasic sites. Oncogene 30:4731-9
Madigan, James P; Bodemann, Brian O; Brady, Donita C et al. (2009) Regulation of Rnd3 localization and function by protein kinase C alpha-mediated phosphorylation. Biochem J 424:153-61
Roberts, Patrick J; Mitin, Natalia; Keller, Patricia J et al. (2008) Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification. J Biol Chem 283:25150-63
Onesto, Cercina; Shutes, Adam; Picard, Virginie et al. (2008) Characterization of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases. Methods Enzymol 439:111-29
Shutes, Adam; Onesto, Cercina; Picard, Virginie et al. (2007) Specificity and mechanism of action of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases. J Biol Chem 282:35666-78
Yohe, Marielle E; Rossman, Kent L; Gardner, Olivia S et al. (2007) Auto-inhibition of the Dbl family protein Tim by an N-terminal helical motif. J Biol Chem 282:13813-23
Loomis, Rebecca J; Holmes, Derek A; Elms, Andrew et al. (2006) Citron kinase, a RhoA effector, enhances HIV-1 virion production by modulating exocytosis. Traffic 7:1643-53

Showing the most recent 10 out of 19 publications