Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research,Utrecht University, Padualaan 8, 3584 CH, Utrecht, THE NETHERLANDS.
The primary focus of this thesis is the functional characterization of the novel A-kinase anchoring protein (AKAP), sphingosine kinase interacting protein (SPHKAP) and its associated type I cAMP-dependent protein kinase holoenzyme (PKA). As an introduction to this topic, Chapter 1 starts with a general overview of cAMP (3’-5’cyclic adenosine monophosphate) signaling at the cellular level. cAMP regulates cellular functions primarily by activating a single kinase, PKA. Therefore, compartmentalization of PKA plays a key role in the spatial and temporal specificity of parallel cAMP signaling events that occur in the same cell. This is mediated through the interaction of PKA’s regulatory subunits (PKA-R) with many different AKAPs. AKAPs often form highly efficient, compact signalosomes by harboring other signaling proteins such as phosphatases and phosphodiesterases alongside PKA. The selective binding of AKAPs to one of the major isoforms of PKA-R (type I (PKA-RI) and PKA-R type II (PKA-RII)) is an important feature of specific cAMP signaling. Therefore, chapter 1 continues to discuss in detail the key differences in structural features between the relatively well characterized PKA-RII and the largely unexplored PKA-RI, which may explain why PKA-RI is more selective towards a small subset of AKAPs and why RII-specific AKAPs are likely more abundantly identified thus far. Then, a systematic overview of all mammalian AKAPs identified thus far is provided. Since SPHKAP was abundantly identified in ventricular tissue, chapter 1 continues with a short functional focus on AKAPs functioning in cardiac physiology and pathophysiology. In the light of SPHKAPs putative role in mitochondrial induced apoptosis (chapter 5), chapter 1 continues with the general basics of apoptosis with a focus on intrinsic or mitochondrial-mediated pathways. The intrinsic pathway proceeds through the coordinated regulation of multiple anti- and pro-apoptotic BCL-2 family proteins that ultimately induce the discontinuity of the outer mitochondrial membrane and the release of cytochrome C. Within the BCL-2 protein family a subclass of pro-apoptotic members termed the BH3-only proteins exists, which directly and/or indirectly regulate other anti- and pro-apoptotic BCL-2 family proteins to engage apoptosis. The focus of this review is to discuss the characteristics of the BH3 domain and the two mutual models by which BH3-only proteins mediate apoptosis.