This proposal tests a new hypothesis that phosphoinositide (PI) 3-kinase synergizes with phospholipase C in initiating receptor-mediated calcium signaling via a phosphatidylinositol 3,4,5-trisphosphate PI(3,4,5) P3]-sensitive Ca2+-entry pathway. This Pl(3,4,5)P3-mediated Ca2+ influx mechanism is unique in many types of hematopoietic cells, and represents a crucial element in PI 3-kinase-mediated signaling cascades that lead to cell activation. Consequently, this novel Ca2+-entry system may serve as a potential target for diseases related to, but not limited to, blood clotting, type I allergy, and T-cell immunity. This hypothesis will be tested in Jurkat T cells, an exquisite model to study PI 3-kinase signal transduction and Ca2+-entry mechanisms.
Specific aim 1 is to establish the obligatory role of PI 3-kinase in receptor-mediated Ca2+ signaling. This will be accomplished by a combined strategy using pharmacological inhibitors and a molecular genetic approach in which Jurkat T cells are stably transfected with a dominant-negative form of the p85 regulatory subunit to suppress the activity of endogenous PI 3-kinase. This strategy allows the assessment of the functional role of PI 3-kinase in Ca2+ regulation following receptor activation. The second specific aim will be to study interactions between PI(3,4,5)P3-mediated Ca2+ entry and other Ca2+ signaling components in the regulation of T-cell Ca2+ homeostasis. Receptor-mediated Ca2+ signaling is a highly complicated event that involves the concerted action of multiple Ca2+-transporting mechanisms in different cellular compartments of T cells. One plausible factor that links these multiple mechanisms is Ca2+ itself. We propose an integrated approach to study the communication of Pl(3,4,5)P3-mediated Ca2+ entry with lP3-mediated Ca2+ release and with store-operated (capacitative) Ca2+ influx. The third specific aim will be to purify and characterize the PI(3,4,5)P3-binding protein in the T-cell plasma membrane. High affinity PI(3,4,5)P3-binding proteins have been demonstrated in T cell plasma membranes using biotinylated PI(3,4,5)P3 as an affinity probe. This affinity ligand in conjunction with a photoaffinity probe permits rapid purification of the PI(3,4,5)P3-binding protein for sequencing and characterization. Sequence homology analysis will provide useful information concerning the identity of the PI(3,4,5)P3-binding protein. Functional characterization will be carried out by examining the effect of affinity-purified proteins on PI(3,4,5)P3- and anti-CD3 mAb-activated Ca2+ influx in Jurkat T cells. These studies will help understand how the PI(3,4,5)P3-binding protein mediates Ca2+ entry across the plasma membrane.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053448-07
Application #
6636150
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Chin, Jean
Project Start
1996-08-01
Project End
2005-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
7
Fiscal Year
2003
Total Cost
$221,250
Indirect Cost
Name
Ohio State University
Department
Type
Schools of Pharmacy
DUNS #
071650709
City
Columbus
State
OH
Country
United States
Zip Code
43210
Tseng, Ping-Hui; Lin, Ho-Pi; Hu, Hongzhen et al. (2004) The canonical transient receptor potential 6 channel as a putative phosphatidylinositol 3,4,5-trisphosphate-sensitive calcium entry system. Biochemistry 43:11701-8
Yang, Chih-Cheng; Lin, Ho-Pi; Chen, Chang-Shi et al. (2003) Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells. J Biol Chem 278:25872-8
Johnson, Amy J; Hsu, Ao-Lin; Lin, Ho-Pi et al. (2002) The cyclo-oxygenase-2 inhibitor celecoxib perturbs intracellular calcium by inhibiting endoplasmic reticulum Ca2+-ATPases: a plausible link with its anti-tumour effect and cardiovascular risks. Biochem J 366:831-7
Johnson, A J; song, X; Hsu, A et al. (2001) Apoptosis signaling pathways mediated by cyclooxygenase-2 inhibitors in prostate cancer cells. Adv Enzyme Regul 41:221-35
Wang, D S; Chen, C S (2001) Synthesis and biological evaluation of L-alpha-phosphatidyl-D-3-deoxy-3-heteromethyl-myo-inositols as phosphoinositide 3-kinase inhibitors. Bioorg Med Chem 9:3165-72
Ching, T T; Hsu, A L; Johnson, A J et al. (2001) Phosphoinositide 3-kinase facilitates antigen-stimulated Ca(2+) influx in RBL-2H3 mast cells via a phosphatidylinositol 3,4,5-trisphosphate-sensitive Ca(2+) entry mechanism. J Biol Chem 276:14814-20
Ching, T T; Lin, H P; Yang, C C et al. (2001) Specific binding of the C-terminal Src homology 2 domain of the p85alpha subunit of phosphoinositide 3-kinase to phosphatidylinositol 3,4,5-trisphosphate. Localization and engineering of the phosphoinositide-binding motif. J Biol Chem 276:43932-8
Wang, D S; Hsu, A L; Chen, C S (2001) A phosphatidylinositol 3,4,5-trisphosphate analogue with low serum protein-binding affinity. Bioorg Med Chem 9:133-9
Hsu, A L; Ching, T T; Wang, D S et al. (2000) The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem 275:11397-403
Hsu, A L; Ching, T T; Sen, G et al. (2000) Novel function of phosphoinositide 3-kinase in T cell Ca2+ signaling. A phosphatidylinositol 3,4,5-trisphosphate-mediated Ca2+ entry mechanism. J Biol Chem 275:16242-50

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