Micah Schott

Date of Award

January 2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Bryon D. Grove

Abstract

A-Kinase Anchoring Proteins (AKAPs) comprise a family of roughly 70 scaffolds that anchor PKA and other enzymes to a variety of subcellular compartments. Although characterized as "anchoring proteins", some AKAPs are not spatially and temporally static but can undergo dynamic subcellular trafficking, thus repositioning anchored enzyme complexes within the cell. Gravin (also called SSeCKS or AKAP12) anchors PKA and other enzymes to the plasma membrane but redistributes to the cytosol upon intracellular calcium ([Ca2+]i) elevation. However, the impact of gravin redistribution on PKA-dependent signaling pathways is poorly understood. We hypothesize that through Ca2+-mediated redistribution, gravin facilitates cross-talk between Ca2+-dependent and PKA-dependent pathways.

First, we tested this by characterizing the impact of [Ca2+]i elevation on the distribution of gravin and PKA. In cells expressing gravin-EGFP, [Ca2+]i elevation with ionomycin or thapsigargin caused gravin redistribution from the cell periphery to the cytosol in as little as 60 seconds. ATP treatment also triggered gravin redistribution through receptor-mediated pathways involving both [Ca2+]i and PKC. Gravin redistribution in response to ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localization at the cell periphery. In addition, we also found that a fourth putative calmodulin binding domain, which we call CB4 (a.a. 669-693), is essential for localization of gravin to the cell periphery. Either deletion of the CB4

domain or mutation of a calmodulin-binding consensus sequence within the CB4 domain disrupted the membrane localization of gravin.

Next, we measured the impact of exogenous gravin-V5/His expression on compartmentalized PKA activity using the PKA FRET biosensor AKAR3. Expression of gravin-V5/His in AN3 CA cells, which lack endogenous gravin, caused an increase in forskolin-stimulated PKA activity at the plasma membrane when compared to control cells lacking gravin. Under these conditions, gravin also decreased PKA activity in the cytosol. Gravin's impact on subcellular PKA activity required both interaction with PKA and localization at the cell periphery. Pre-treatment with the [Ca2+]i elevating agent thapsigargin caused gravin redistribution and inhibited gravin-mediated elevation of PKA activity the plasma membrane. These results support the hypothesis that gravin mediates crosstalk between Ca2+-dependent and PKA-dependent signaling pathways.

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