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Calcium-dependent protein kinase-A-mediated regulation of the plasma membrane calcium pump

Reference	BB/C514607/1
Principal Investigator / Supervisor	Dr Jason Bruce
Co-Investigators / Co-Supervisors
Institution	The University of Manchester
Department	Life Sciences
Funding type	Research
Value (£)	253,316
Status	Completed
Type	Research Grant
Start date	01/06/2005
End date	30/09/2008
Duration	40 months

Abstract

The spatio-temporal shaping of intracellular calcium ([Ca2+]i) signalling encodes numerous functional responses as diverse as regulation of vascular tone, neuronal plasticity and secretion. The specificity of these [Ca2+]i signals can be achieved by activating other signalling pathways, such as cAMP and PKA, which modulates the activity of key Ca2+ transport proteins, such as inositol 1,4,5-trisphosphate receptors (insP3R) or plasma membrane Ca2+-ATPase(PMCA). The molecular mechanism of such signalling cross-talk is critical to our understanding of the specificity of stimulus-response coupling in all cells. The PMCA is important for maintaining low resting [Ca2+]i and is also dynamically regulated by Ca2+ in an integrative manner, important for modulating [Ca2+]i oscillations. In addition, the PMCA contains a PDZ (PSD-95/Dlg/ZO-1)-binding domain, that binds to multi-protein signalling complexes, important for regulating local [Ca2+]i signals and thus local Ca1+-dependent effectors. cAMP-mediated regulation of the PMCA is poorly understood. However, I have recently demonstrated in parotid acinar cells that PKA potentiates and phosphorylates the PMCA but only in the presence of [Ca2+]i -raising agents. In addition, preliminary data shows that the PMCA binds to the accessory proteins, ezrin, EBP50 and PKA, which is also Ca2+-dependent. Therefore, the overall objective of the current proposal is to determine the specific molecular mechanism for this Ca2+-dependent, PKA-mediated regulation of the PMCA by addressing two separately testable hypotheses that: (1) a Ca2+-mediated conformational change in the PMCA exposes an otherwise cryptic PKA phosphorylation site, or (II) a Ca2+-mediated assembly of a signalling complex brings PKA closer to the PMCA allowing targeted regulation in effect, a Ca2+-dependent molecular machine. This will be achieved by first identifying the specific PMCA isoform phosphorylated by PKA in parotid acinar cells. Using recombinant DNA technology the candidate PMCA isoform will be transiently over-expressed in a heterologous system and PMCA activity assayed by measuring [Ca2+]i clearance under conditions where Ca2+ efflux is isolated in intact cells. The effect of PKA on PMCA activity will be compared between cells over-expressing wild-type (WT)-PMCA versus mutant-PMCA (PKA site or CaM-binding mutations), truncated PMCA (lacking the PDZ-binding domain) or modified CaM. Finally, yeast-two-hybrid screens and GST-pull-down assays will identify PMCA-binding proteins and investigate the nature of the Ca2+-dependent molecular machine. Data from these studies will elevate the importance of the PMCA from a simple housekeeping role to one that can be dynamically regulated by Ca2+ and cAMP, important for shaping [Ca2+]i signals and thus fine-tuning of Ca2+-dependent effectors, mechanisms that have been largely ignored in the past. The current proposal will greatly enhance our understanding of this molecular mechanism and begin to unravel the complexity and increasingly important role of the PMCA in stimulus-response coupling.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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