Online

Sheehe, Jessica Lynne

Cellular, Molecular and Biomedical Sciences Graduate Program

2018

Ph. D.

The type I[alpha] cGMP-dependent protein kinase (PKG I[alpha]) is an essential regulator of vascular tone and systemic blood pressure. Located in the smooth muscle of resistance vessels, PKG I[alpha] stimulates vasodilation through the phosphorylation of multiple intracellular substrates. Its primary regulator is the small molecule, 3',5'-cyclic guanosine monophosphate (cGMP); however, the I[alpha] isoform can also be activated by oxidation. Despite the established physiological importance of PKG I[alpha], 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 I[alpha] activation. Using a monomeric, N-terminally truncated form of PKG I[alpha] ([deltal]53), 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 I[alpha]. Using PKG I[alpha] 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 I[alpha] 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.