Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10815453 | Cellular Signalling | 2013 | 11 Pages |
Abstract
A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300Â kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca2Â +]i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin-EGFP revealed that Ca2Â + elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60Â s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca2Â +]i elevation and PKC activation. To understand the mechanism for Ca2Â + mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670-694) is critical for targeting gravin to the cell cortex despite its location downstream of gravin's membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin-EYFP and PKA RII-ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca2Â + and PKC.
Keywords
ECFPbisindolylmaleimideionomycinβ2ARSOCEEYFPThapsigarginPDE4inositol triphosphatePKCRIIAKAPPLCInsP3AKAP12GPCRpKaSSeCKSCB4eGFP1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)sarco/endoplasmic reticulum calcium ATPaseG protein coupled receptor[Ca2 +]iAdenosine TriphosphateATPSESenhanced cyan fluorescent proteinBAPTA-AMCAMintracellular calcium concentrationphosphodiesterase type 4phospholipase CSERCABIMStore-operated calcium entryA-kinase anchoring proteinenhanced yellow fluorescent proteinenhanced green fluorescent proteinprotein kinase AProtein kinase CCalciumGravinβ2-adrenergic receptorpurinergic receptor
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Micah B. Schott, Bryon Grove,