Isoform-specific Regulation Mechanisms of cGMP-dependent Protein Kinases (PKGs)

The cGMP-dependent protein kinase is an important player in several vital pathways. The PKG I splice variants (Iα and Iβ) are essential for the NO/cGMP-dependent relaxation of vascular smooth muscles, while the PKG II isoform plays a pivotal role in skeletal homeostasis and intestinal chloride secretion. Furthermore, pathologically increased cGMP levels in different forms of retinal degeneration (RD) imply PKG as a potential perpetrator in photoreceptor cell death. Activation of PKG isoenzymes upon cGMP binding to the cyclic nucleotide binding domains (CNBD) has been an object of research for the past decades. Reported values vary depending on the experimental set-up and employed PKG constructs. This thesis aims to investigate PKG isoenzyme activation under identical conditions to unravel peculiar characteristics of each isoform. CNBD binding-deficient mutants were utilized to examine the role of the CNB-A and CNB-B in PKG isoform-specific activation in detail. The data shown here reveal key differences in the activation mechanism of each PKG. While CNB-A:cyclic nucleotide binding is the driving force for PKG Iα and PKG II activation, simultaneous CNB-A and CNB-B binding is a prerequisite for PKG Iβ kinase activation. SPR binding data in the presence of the cofactors ATP and Mg2+ and the peptide substrate VASPtide show isoform-specific effects on cGMP binding. ATP and MgCl2 reduced the affinity of cGMP to PKG Iα and PKG II. The addition of VASPtide increased the affinity of cGMP to values comparable to the activation constants. This is contrasted by PKG Iβ, where ATP and Mg2+ did not alter its transient interaction with cGMP; however, VASPtide again increased cGMP affinities. In the context of retinal degeneration, unique interaction profiles between each PKG isoform and antagonistic cGMP analogues were recorded in a spectrophotometric kinase assay, revealing characteristic differences between the PKG I splice variants. Furthermore, RNAscope was utilized to identify PKG isoforms expressed in the murine retina, as immunohistochemistry failed to discriminate between the PKG I splice variants. These experiments specified PKG Iβ and PKG II as the isoforms most likely expressed in photoreceptor cells. Based on these interaction studies and RNAscope, Rp-8-Br-PET-cGMPS (CN003), Rp-8-Br-pMe-PET-cGMPS (CN238), Rp-8-oAPT-cGMPS (CN229), and Rp-8-oAPT-PET-cGMPS (CN230) were identified as the most promising analogues. In-depth analysis revealed that their antagonistic potential is primarily driven by CNB-A binding and is not affected by the cofactors ATP and MgCl2, alluding to their cGMP competitive potential in vivo. Structural and biochemical data presented in this thesis enable the generation of detailed activation models for all human PKG isoforms, thus providing a clearer mechanistic understanding to aid the development of cGMP analogue-driven therapeutic approaches.