Identification and Structural Characterization of Novel A-kinase Anchoring Proteins

Abstract

The work described in this thesis initially focusses on the discovery of a novel PKA-RI specific AKAP: small membrane AKAP (smAKAP). Afterwards we centre on the structural interaction between smAKAP and PKA-RI to reveal the first PKA-RI specific AKAP bound to PKA-RI crystal structure. Interestingly, a novel self-inhibition mechanism was discovered which allows PKA to block its binding to AKAPs under certain restrictions. In order to fully understand the specific limitations associated with binding a study centering on the PKA-RI and PKA-RII interactions with AKAPs was performed. In Chapter 2, recent literature on the structural interface between PKA-RI/RII and AKAPs is reviewed. Most of these structural studies involve either X-ray crystallography, three-dimensional NMR, binding affinity assays and various other biochemical methods. In Chapter 3, the discovery and initial characterization of a novel AKAP termed smAKAP is described. Via binding affinity assays and imaging techniques it is shown that smAKAP is PKA-RI specific. The intracellular location of smAKAP at the plasma membrane is shown by means of fluorescence imaging and advanced electron microscopy. In Chapter 4, structural techniques such as hydrogen/deuterium exchange and X-ray crystallography are applied to probe the interaction interface between smAKAP and PKA-RI. Additionally, via a phosphoproteomics study it was shown that in the middle of the A-kinase binding domain of smAKAP there is a putative PKA phosphorylation site. Upon phosphorylation of this site, PKA cannot bind to smAKAP anymore. A mechanistic model on how this disruption occurs is presented. In Chapter 5, a novel bioinformatic tool, THAHIT (THe AKAP/amphipathic Helix Identification Tool), is able to predict PKA-RIα and/or PKA-RIIα binding domains. This software package is based on currently known and well-established PKA-RIα and PKA-RIIα binding motifs. After applying it on all known AKAPs, numerous new PKA-RIα and PKA-RIIα binding domains in these AKAPs were found and/or narrowed down. Several of these were confirmed via conservation (BlastP), in silico docking studies using HADDOCK and in vitro binding studies using fluorescence anisotropy. In addition, several cAMP pull-downs were investigated for potential novel AKAPs using THAHIT. Here we propose a novel very large AKAP: vlAKAP