The type Iα cGMP-dependent protein kinase (PKG Iα) is an essential regulator of vascular tone and systemic blood pressure. Located in the smooth muscle of resistance vessels, PKG Iα stimulates vasodilation through the phosphorylation of multiple intracellular substrates. Its primary regulator is the small molecule, 3',5'-cyclic guanosine monophosphate (cGMP); however, the Iα isoform can also be activated by oxidation. Despite the established physiological importance of PKG Iα, the structural underpinnings of these two activation mechanisms are largely unknown. The work presented in this dissertation demonstrates the importance of the cGMP-binding domain A (CBD-A) in regulating both of these mechanisms of PKG Iα activation. Using a monomeric, N-terminally truncated form of PKG Iα (Δ53), Chapter 2 investigates the mechanism of inhibition through the autoinhibitory domain and the influence of dimerization on cooperative cGMP-dependent activation and cyclic nucleotide selectivity. We observed that autoinhibition occurs in cis, whereas cooperativity requires interprotomer contacts facilitated by the N-terminal dimerization domain. Furthermore, the loss of selectivity for cGMP over cAMP of this construct suggests the dimerization domain plays a critical role in preventing cross-reactivity with cAMP-dependent signaling. These observations culminate into an overarching model wherein binding of cGMP to CBD-A is necessary and sufficient for activation and cooperativity is driven by the dimerization domain. Chapter 3 investigates the cysteine residues that mediate oxidation-dependent activation of PKG Iα. Using PKG Iα constructs with point mutations at specific cysteine residues, it was found that oxidation-dependent activation is driven by C117 in CBD-A. Furthermore, the interprotomer disulfide bond that forms in the dimerization domain at C42 does not contribute to this mechanism. Finally, we propose a model wherein the disulfide bond that forms between C117 and the adjacent cysteine at position 195 acts as a protective mechanism to prevent activation and higher oxidation states form contacts with nearby residues in the linker region of PKG Iα to disrupt binding of the adjacent autoinhibitory domain to the catalytic domain. Finally, Chapter 4 provides a discussion of the results presented herein in context with previous studies and suggests future directions for the PKG field.

cGMP-dependent protein kinases (PKG) are key players in intracellular second messenger signaling within many cellular systems throughout the body. Most notably PKG is known for its role in smooth muscle relaxation (Pfeiffer et.al, 1999). The Iα PKG isozyme has been identified as the primary effector of the nitric oxide pathway (and serves to be a novel drug target). To date the overall knowledge of structure and function of PKG is lacking in terms of the mechanisms of activation and the structural orientations that coordinate them. Recently, our laboratory has solved the crystal structure of the regulatory domain of PKG Iα, which revealed a previously unknown α-helical domain dubbed the Switch Helix (SW) (Osborne et.al, 2011). The SW domain was found to be a site of interprotomer communication via hydrophobic interactions between its C-terminus and hydrophobic residues, named the nest located on the opposing protomer. Synthetic peptides derived from the SW domain, named S-tides, dosedependently activate PKG Iα (Moon et.al, 2015). In addition, the amino acid residues of the nest are in proximity to the cGMP binding site B. It was hypothesized that the binding site for S-tides (nest) and the cGMP binding site B interact and are co-dependent of one another. The hypothesis of this thesis is the binding site for the S-tides (nest) and the cGMP binding site B interact and are co-dependent of one another. To test this hypothesis two aims were constructed: Aim 1: To develop an S1.5 analog that utilizes both the nest and the B-site to increase S-tide activity, Aim 2: To explore the intricacies of these modes of activation and how they interact with each other to obtain a better understanding of the interplay between these two sites. First, based on the most potent S-tide S1.5 (YEDAEAKAKYEAEAAFFANLKLSD, Ka=6 μM), two analogs were synthesized. The peptide S2.5 which lacked the amino acids LSD at the C-terminus showed a three-fold lower activation constant (Ka= 15 μM), although the molecule retained its helicity as demonstrated by circular dichroism. The second analog, S3.5 contained unnatural amino acid components from a molecular modeling approach in an effort to further increase the affinity by interacting with the adjacent cGMP binding site B. However, S3.5 showed further reduction in activity with an activation constant of 70 μM. These findings led us to conclude that the failure of the SAR approach indicates a different mode of S-tide activation as had been previously thought. Next, we investigated the role of the cGMP binding site B in the mechanism of S-tide mediated PKG Iα activation. Co-activation assays with cGMP and S1.5 demonstrated that cGMP activation is not altered in the presence of S1.5. Furthermore, S1.5 mediated activation is negatively affected in the presence of cGMP. These results suggest that the B-site of cGMP does not positively enforce the S1.5 activation kinetics. Next, we employed the PKG Iα mutant E292A, which cannot bind cGMP to the B-site (Moon et.al., 2018). Interestingly, this mutant retains the activation kinetics of PKG Iα WT when activated via S1.5 and cGMP. Thus, the cGMP binding site B is not crucial in the activation mechanisms of activating PKG Iα with cGMP. Likewise, the cGMP binding site B is not crucial in the activation mechanisms of activating PKG Iα with S1.5. To further support these findings, the PKG Iα mutant C42A, which showed crippled cGMP activation kinetics could be activated with S1.5 with a potency similar to wild type. Taken together, the results in this thesis demonstrate that in contrast to the initial hypothesis the binding sites for S-tides and cGMP, although in proximity, show no experimental support of a positive interaction. These findings are significant as they reveal that S1.5 mediated activation of PKG is truly independent of cGMP, thereby providing a molecular platform for the therapeutic development of these unique peptides.

<p>Malignant primary brain tumors, gliomas, often overexpress both platelet-derived growth factor (PDGF) ligands and receptors providing an autocrine and/or paracrine boost to tumor growth. Glioblastoma multiforme (GBM) is the most frequent glioma. Its aggressive and infiltrative growth renders it extremely difficult to treat. Median survival after diagnosis is currently only 14 months. </p><p>The present thesis describes the use of retroviral tagging to identify candidate cancer-causing genes that cooperate with PDGF in brain tumor formation. Newborn mice were injected intracerebrally with a Moloney murine leukemia retrovirus carrying the <i>sis</i>/PDGF-B oncogene and a replication competent helper virus. Brain tumors with many characteristics of human glioblastomas developed after 13-42 weeks. </p><p>Analysis of proviral integrations in the brain tumors identified almost 70 common insertion sites (CISs). These CISs were named brain tumor loci and harbored known but also putative novel cancer-causing genes.</p><p>An array with over 15000 unique cDNAs was used to screen for differentially expressed genes in the mouse brain tumors compared to normal brain. Known tumor genes and markers of immature cells were upregulated in the tumors. Short latency tumors were further distinguished as fast growing and GBM-like. Long latency tumors resembled slow-growing oligodendrogliomas and contained significantly less integrations as compared to short latency tumors.</p><p>The gene <i>Prkg2</i>, encoding the cGMP-dependent protein kinase II, was targeted by insertions in two brain tumors. Overexpression of <i>Prkg2</i> in human glioma cell lines led to a reduction in colony formation, cell proliferation and migration. A glioma cell line expressing markers of immature stem cells showed loss of cell adhesion, G1 cell cycle arrest and decreased activation of the survival signaling protein Akt upon stimulation with a cGMP analog that activates the <i>Prkg2</i> protein. The present thesis shows that proviral tagging may be a useful tool in the search for candidate glioma genes.</p>

Malignant primary brain tumors, gliomas, often overexpress both platelet-derived growth factor (PDGF) ligands and receptors providing an autocrine and/or paracrine boost to tumor growth. Glioblastoma multiforme (GBM) is the most frequent glioma. Its aggressive and infiltrative growth renders it extremely difficult to treat. Median survival after diagnosis is currently only 14 months. The present thesis describes the use of retroviral tagging to identify candidate cancer-causing genes that cooperate with PDGF in brain tumor formation. Newborn mice were injected intracerebrally with a Moloney murine leukemia retrovirus carrying the sis/PDGF-B oncogene and a replication competent helper virus. Brain tumors with many characteristics of human glioblastomas developed after 13-42 weeks. Analysis of proviral integrations in the brain tumors identified almost 70 common insertion sites (CISs). These CISs were named brain tumor loci and harbored known but also putative novel cancer-causing genes. An array with over 15000 unique cDNAs was used to screen for differentially expressed genes in the mouse brain tumors compared to normal brain. Known tumor genes and markers of immature cells were upregulated in the tumors. Short latency tumors were further distinguished as fast growing and GBM-like. Long latency tumors resembled slow-growing oligodendrogliomas and contained significantly less integrations as compared to short latency tumors. The gene Prkg2, encoding the cGMP-dependent protein kinase II, was targeted by insertions in two brain tumors. Overexpression of Prkg2 in human glioma cell lines led to a reduction in colony formation, cell proliferation and migration. A glioma cell line expressing markers of immature stem cells showed loss of cell adhesion, G1 cell cycle arrest and decreased activation of the survival signaling protein Akt upon stimulation with a cGMP analog that activates the Prkg2 protein. The present thesis shows that proviral tagging may be a useful tool in the search for candidate glioma genes.

Phosphodiesterase Type 5 (PDE5) inhibitors are cardioprotective against ischemia/reperfusion (I/R) injury. However, it remains uncertain if I/R affects PDE5. We hypothesized that generation of reactive oxygen species (ROS) during I/R leads to upregulation of PDE5, which contributes to pathological changes following acute myocardial infarction (AMI). Adult male ICR mice were subjected to 30 minutes of in vivo or ex vivo I/R. To examine the role of ROS, a subset of hearts were perfused with 100 µM hydrogen peroxide (H2O2). Expression and activity of PDE5, pPDE5, and cGMP-dependent protein kinase (PKG) were measured by Western blots and spectrophotometric assay. The results show that ischemia and I/R significantly increased expression of PDE5. H2O2 had no effect on PDE5 expression and activity but significantly increased PKG activity. We conclude that acute cardiac ischemia or I/R upregulate PDE5 independent of oxidant stress in the heart.