Rapamycin is a macrolide natural product with multiple clinical applications. Rapamycin serves as an immunosuppressant following organ transplantation, as chemotherapy for tumors and as an inhibitor of restenosis of arteries. Yet, the mechanism of action of rapamycin as an antiproliferative agent remains incompletely understood and resistance to its effects undermines the clinical applications. Rapamycin binds to a ubiquitous intracellular receptor protein called FKBP12 and inhibits the protein kinase Target Of Rapamycin (TOR), a member of the family of cell cycle checkpoint kinases. TOR is conserved among all eucaryotes and functions in control cell growth and cell proliferation. Yeast genetics has revealed that signaling downstream of TOR requires the essential protein Ser/Thr phosphatase SIT4 (mammalian PP6), which controls induction of G1 cyclins and cell cycle progression. In addition, the essential yeast protein TAP42 and recently found TIP41 regulate the SIT4 phosphatase and possibly also the PP2A phosphatase in yeast. This revised continuation application proposes four Specific Aims: 1) Elucidate the mechanism for rapamycin and TOR regulation of protein phosphatases PP6 and PP2A involving phosphorylation and/or subunit interchange. 2) Define the structural determinants required for interaction between human alpha-4 (TAP42) and its binding partner AlP (TIP41) using transient expression of truncated and mutated proteins and co-precipitation. 3) Determine the function of the unique N terminus of PP6 (SIT4) that confers the ability of this phosphatase to promote G1 to S phase progression by producing fusion proteins and chimeric phosphatases to test for specific localization, dominant negative interference or change in catalytic properties. 4) Establish that cellular resistance to rapamycin depends on availability of alpha-4 to associate with PP6 (SIT4) phosphatase by increasing and depleting cellular levels of AlP and alpha-4 using transfection and siRNA knock-down. The proposed studies will give new insights into an under-studied phosphatase-signaling pathway and provide information on the molecular basis for action of and resistance to rapamycin.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA077584-09
Application #
7256410
Study Section
Biochemistry Study Section (BIO)
Program Officer
Mufson, R Allan
Project Start
1999-04-01
Project End
2008-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
9
Fiscal Year
2007
Total Cost
$213,076
Indirect Cost
Name
University of Virginia
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Molhoek, Kerrington R; Shada, Amber L; Smolkin, Mark et al. (2011) Comprehensive analysis of receptor tyrosine kinase activation in human melanomas reveals autocrine signaling through IGF-1R. Melanoma Res 21:274-84
Molhoek, Kerrington R; McSkimming, Chantel C; Olson, Walter C et al. (2009) Apoptosis of CD4(+)CD25(high) T cells in response to Sirolimus requires activation of T cell receptor and is modulated by IL-2. Cancer Immunol Immunother 58:867-76
Guergnon, Julien; Derewenda, Urszula; Edelson, Jessica R et al. (2009) Mapping of protein phosphatase-6 association with its SAPS domain regulatory subunit using a model of helical repeats. BMC Biochem 10:24
Prickett, Todd D; Ninomiya-Tsuji, Jun; Broglie, Peter et al. (2008) TAB4 stimulates TAK1-TAB1 phosphorylation and binds polyubiquitin to direct signaling to NF-kappaB. J Biol Chem 283:19245-54
Molhoek, Kerrington R; Griesemann, Heinrich; Shu, Jianfen et al. (2008) Human melanoma cytolysis by combined inhibition of mammalian target of rapamycin and vascular endothelial growth factor/vascular endothelial growth factor receptor-2. Cancer Res 68:4392-7
Stefansson, Bjarki; Ohama, Takashi; Daugherty, Abbi E et al. (2008) Protein phosphatase 6 regulatory subunits composed of ankyrin repeat domains. Biochemistry 47:1442-51
Yoo, Sunny J-S; Jimenez, Rosa H; Sanders, Jennifer A et al. (2008) The alpha4-containing form of protein phosphatase 2A in liver and hepatic cells. J Cell Biochem 105:290-300
Stefansson, Bjarki; Brautigan, David L (2007) Protein phosphatase PP6 N terminal domain restricts G1 to S phase progression in human cancer cells. Cell Cycle 6:1386-92
Yoo, Sunny J-S; Boylan, Joan M; Brautigan, David L et al. (2007) Subunit composition and developmental regulation of hepatic protein phosphatase 2A (PP2A). Arch Biochem Biophys 461:186-93
Yang, Jing; Roe, S Mark; Prickett, Todd D et al. (2007) The structure of Tap42/alpha4 reveals a tetratricopeptide repeat-like fold and provides insights into PP2A regulation. Biochemistry 46:8807-15

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