A multi-angular mass spectrometric view at cyclic nucleotide signaling proteins : Structure/function and protein interactions of cAMP- and cGMP-dependent protein kinase

The primary focus of this thesis is the two kinases PKA and PKG, cAMP and cGMP dependent protein kinase respectively. PKA and PKG are studied both at structure/function level as well as at the level of interaction with other proteins in tissue. Our primary methods are all based on mass spectrometry. First, a brief overview of molecular signaling events involved in the function of the cardiovascular system, especially those involving the cyclic nucleotides cAMP (cyclic adenosine monophosphate) and cGMP (cyclic guanosine monophosphate) is given. Besides the specific functions of the two kinases PKG and PKA, other proteins involved in cyclic nucleotide signaling, like phosphodiesterases, cyclic nucleotide activated ion channels and Epac are briefly discussed. Special attention is given to compartmentalized signaling events that involve scaffolding proteins, for PKA and PKG designated as AKAPs and GKAPs. The structure function relationships in PKA and PKG are also described. Specifically, mass spectrometric techniques that were used in structure/function studies are described in somewhat more detail. The structure function relationships of PKG is the focus of chapter 2 and 3. Chapter 2 studies this by intrinsic fluorescence stability measurements and limited proteolysis to show that the stability of PKG is mediated by a single amino acid that acts as a stability switch. In chapter 3, a lysine surface labeling technique was developed and tested on the E. Coli Colicin E9-Im9 protein complex. Then it was applied to elucidate the differential solvent exposure of PKG's different domains upon cGMP binding, which eventually proved more challenging than expected. This is either due to the fact that no lysines are in direct proximity of the affected sites, or that affected sites all reside in the uncovered 40% of the protein sequence. It is also possible that the basal kinase activation of PKG takes place on a short timescale, thereby eliminating differences. The next two chapters of this thesis focus on the use and development of chemical proteomic approaches in the search for (novel) PKA AKAP signaling complexes involved in heart function. Chapter 4 describes the initial development of a chemical proteomics method to selectively enrich PKA/AKAP, and PKG, signaling protein complexes from rat ventricular tissue by using immobilized cAMP or cGMP. Among the proteins enriched for, several different AKAPs not earlier described in heart tissue were observed. Most interesting was the observation that the protein SKIP (Sphingosine Kinase type 1 Interacting Protein), earlier described as an anchor for sphingosine kinase-1, could be assigned as a novel AKAP. Chapter 5 continues with the application of the cyclic nucleotide pull down protocol. A more detailed relative quantification procedure is described as well to more reliably allocating proteins to a specific eluting fraction. Attention is focused on the discovery of a novel splice isoform of AKAP2: PALM2-AKAP2, as well as on the observed differential phosphorylation patterns of PKA regulatory subunit type Iα. Finally, all results are placed into perspective in a concluding summary and outlook