3,5-BISTRIFLUOROMETHYL PYRAZOLE (BTP) COMPOUNDS AND REGULATION OF STORE-OPERATED CALCIUM CHANNELS BY THE ACTIN-BINDING PROTEIN DREBRIN
Open Access
- Author:
- Mercer, Jason Chance
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 04, 2005
- Committee Members:
- Avery August, Committee Chair/Co-Chair
Pamela Hankey Giblin, Committee Member
Richard John Frisque, Committee Member
Andrew Thomas Henderson, Committee Member
Blake R Peterson, Committee Member - Keywords:
- calcium
store-operated channel
CRAC
BTP
actin
cytoskeleton
drug - Abstract:
- Stimulation of tyrosine kinase coupled receptors, such as the T cell receptor in T lymphocytes, results in activation of phospholipase-Cγ (PLCγ) which hydrolyzes phosphoinositol 4,5, bisphosphate (PIP2) to generate the second messengers inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG). Similarly, IP3 and DAG are generated by phospholipase-Cβ (PLCβ) downstream of G-protein coupled receptors in other cell types. This results in increased intracellular calcium concentration [Ca2+]i due to the action of IP3 on IP3 receptors (IP3R) in the endoplasmic reticulum (ER) membrane which, when activated, stimulate the release of ER calcium stores into the cytoplasm. Emptying of the ER calcium stores stimulates entry of extracellular calcium through store-operated channels (SOCs), thus maintaining the higher concentration of intracellular Ca2+. [Ca2+]i increases play a critical role in a variety of cellular processes such as transcription factor activation and cytoskeletal reorganization. Although very little is known about the regulation of SOCs, actin cytoskeletal changes have been suggested to be essential for their operation. However, actin cytoskeletal changes appear to be dispensable for ER calcium release. A recent model for activation of SOCS has been proposed wherein the signal between the ER and the plasma membrane that activates SOCs involves a secretion-like mechanism that is blocked by thick cortical actin. Recently, a class of compounds called BTPs (3,5-bistrifluoromethyl pyrazoles) was found to inhibit activation of the calcium regulated transcription factor Nuclear Factor of Activated T cells . We have determined that BTP acts by blocking store-operated calcium entry following Ca2+ store depletion by ionomycin in Jurkat T cells. We have identified the actin reorganizing protein drebrin as a likely target of BTP. We demonstrate that drebrin expression is essential for activation of store-operated calcium entry in Jurkat T cells as reduction in drebrin expression by siRNA treatment results in a block in SOC mediated [Ca2+]i increase but not ER Ca2+ release, similar to BTP treatment. Additionally, we show that BTP is able to block drebrin dependent actin rearrangement. Based on our findings, we propose that BTP blocks SOC activation by preventing drebrin mediated cytoskeletal changes that are necessary for activation of store-operated channels.