Date of Completion

7-1-2013

Embargo Period

6-26-2015

Major Advisor

Dr. Kimberly Dodge-Kafka

Associate Advisor

Dr. Betty Eipper

Associate Advisor

Dr. Bing Hao

Associate Advisor

Dr. Asis Das

Associate Advisor

Dr. Lixia Yue

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The thesis work presented here focuses on AKAP18γ mediated multivalent complex formation and its oligomerization. AKAP18γ belongs to a family of structurally diverse but functionally analogous scaffolding proteins called A-Kinase Anchoring Proteins (AKAPs). These scaffolds specifically target Protein Kinase A (PKA) holoenzyme to a distinct cellular compartment to regulate PKA activity and substrate specificity. Previously, the long isoforms of an AKAP called AKAP18 (AKAP18γ/δ) were established as important cardiac calcium regulating proteins due to their ability to mediate phosphorylation of a protein called Phospholamban (PLN). My dissertation work shows that AKAP18γ directly interacts with Inhibitior-1 (I-1) and enhances its phosphorylation under stimulatory conditions. The AKAP18γ-PKA-I-1 complex together regulates Protein Phosphatase 1 (PP1), also found to be associated with this complex. PP1 is a negative regulator of cardiac function due to its ability to dephosphorylate PLN. Based on these results, we propose a model in which AKAP18γ acts as multivalent complex organizing scaffold that may be targeted to the sarcoplasmic reticulum in cardiac myocytes for PLN phosphorylation regulation. This complex may be found in other cell types to regulate PP1 function alone as I-1, PP1 and AKAP18γ have been found in other tissue.

Moreover, I also discovered that AKAP18γ is an oligomerizing scaffold and the interaction is isoform specific. A shorter isoform of this gene, AKAP18α does not oligomerize and there is no hetero oligomerization between these isoforms (AKA18α and AKAP18γ). Live cell microscopy shows that AKAP18γ can form heterogenous oligomers ranging from monomers to tetramers in cells. Furthermore, in order to understand the physiological implication of this oligomerization, we developed a computational model. The model was based on activation and release of PKA catalytic subunit, which requires PKA regulatory subunit phosphorylation. Presence of multiple PKA holoezymes will potentiate release of multiple PKA catalytic subunits due to increased PKA regulatory subunit phosphorylation. When this possibility was tested against a generic membrane compartmentalized substrate, the computational model revealed increased PKA mediated phosphorylation of the substrate. These results are suggestive of a mechanism in which AKAP18γ oligomerizes to mediate enhanced substrate phosphorylation by PKA.

Based on the evidence presented in this thesis and work done by others, it appears that AKAP18γ not only regulates PLN phosphorylation but also its de-phosphorylation by regulating PP1 activity. Importantly, AKAP18γ can oligomerize which may be the potential mechanism by which AKAP18γ concentrates enzymes required for PLN regulation. The title of the thesis “AKAP18: bidirectional regulator of protein phosphorylation” reflects these properties of AKAP18γ.

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