rosothiols (PSNO), as well as various disulfides (PSSR; Reddie and Carroll, 2008).
Disulfides can be formed within a protein (intradisulfide), between protein subunits (interdisulfide), with low molecular thiol-containing molecules such as glutathione (Sglutathionylation) or hydrogen sulfide (sulfhydration or sulfation; Rudyk and Eaton, 2014). Reversible protein modifications such as disulfide formation can be analogous to well-established posttranslational modifications such as phosphorylation (Schroder and Eaton, 2008). Disulfide bond formation in proteins is a widely recognized cysteine modification. It can influence catalytic activity (Cremers and Jakob, 2013), protein-protein interactions (Banky et al., 2003) and subcellular localization (Brennan et al., 2006). Of course disulfide bonds also play crucial roles in maintaining the structural integrity and correct folding of many proteins (Betz, 1993). Redox proteomic studies searching for proteins that form disulfide bond have shown that a multitude of proteins involved in wide-ranging biological processes including redox homeostasis, chaperone activity, metabolism, transcriptional regulation, and protein translation (Leichert et al., 2008;Reddie and Carroll, 2008;Paulsen and Carroll, 2010) are potentially regulated in this way. When disulfides form in signaling proteins such as phosphatases or kinases, this allows changes in redox state to be integrated with regulation involving protein phosphorylation.
A broad range of protein phosphatases can be regulated by modulation of their thiol redox state, such as low molecular weight protein tyrosine phosphatase (LMW-PTP), phosphatase and tensin homolog (PTEN), cell division cycle dual-specificity phosphatase (Cdc25), protein tyrosine phosphatase 1B (PTP1B), protein tyrosine phosphatase 2α (PTP2α), Src homology region two domain-containing phosphatase-1 and -2 (SHP-1/2), (Salmeen and Barford, 2005;Chen et al., 2009;Paulsen and Carroll, 2010;Tanner et al., 2011). In the case of PTP, the reason disulfide or other modes of oxidation are inhibitory to their activity is because they have a catalytic thiolate that is integral to the dephosphorylation. This is because the thiolate enables a nucleophilic attack on its phospho-tyrosine substrates (Tonks, 2006). A number of kinases have also been shown to be redox regulated, including stress-activated MAPK/thioredoxin peroxidase 1(Sty1/Tpx1), Src tyrosine kinase, apoptosis signalregulated kinase-1 (ASK1; Paulsen and Carroll, 2010). Such redox regulation can also involve disulfide bond formation as occurs with protein kinase A RIα (Brennan et al., 2006), and the focus of this review, namely cGMP-dependent protein kinase-also known as protein kinase G (PKG; Burgoyne et al., 2007).

PKG-isoforms, Structure, and Activation
PKG is a member of the serine/threonine kinase family. Mammals have two PKG genes, prkg1 and prkg2, that encode PKG type I and type II, respectively. PKG I and PKG II are homodimers of two identical subunits (≈75 or ≈85 kDa, respectively) and have similar domain architecture. PKG contains three functional domains (Francis et al., 2010; Figure 1).
The N-terminal domain contains an α-helix with a hydrophobic leucine/isoleucine zipper motif that is responsible for the basal dimerisation of the kinase and its interaction with socalled G-kinase anchoring proteins (GKAPs; Scholten et al., 2008). The regulatory domain on each subunit contains one high affinity and one low affinity cyclic guanosine monophosphate (cGMP) binding site. The catalytic domain, consisting of an ATP/Mg 2+ and a substrate binding site, catalyzes the transfer of the γ phosphate of ATP to the hydroxyl group of a serine/threonine side chain of the target protein. Binding of cGMP to both cGMP binding domains induces a conformational change that relieves the inhibition of the catalytic core by the N-terminus and allows the phosphorylation of substrate proteins (Feil et al., 2003;Scholten et al., 2008). The N-terminus (approximately amino acids 1-100) of PKG I is encoded by two alternatively used exons resulting in the production of two PKG I isoforms, namely PKG Iα and PKG Iβ. Although PKG Iα and Iβ do not differ much in sequence beyond the N-terminus, PKG Iα has more than 10-fold higher affinity for cGMP than PKG Iβ (Ruth et al., 1991;Lee et al., 2011).
PKG isoforms also differ in their tissue and cellular distributions. PKG I is predominantly localized in the cytoplasm, whereas PKG II is typically anchored to the plasma membrane by N-terminal myristoylation (Vaandrager et al., 1996). The PKG Iα isozyme is mainly found in lung, heart, dorsal root ganglia, and cerebellum. In contrast, the Iβ isozyme is highly expressed in platelets, as well as hippocampal and olfactory bulb neurons.
Smooth muscle cells, for example, within the uterus, blood vessels, intestine or the trachea, contain both Iα and Iβ isozymes (Geiselhoringer et al., 2004). PKG II was found mainly in kidney, cerebellum and mucosa (Jarchau et al., 1994).
PKG Iα contains 11 cysteine residues (Takio et al., 1984), five of which have been suggested to contribute to oxidationinduced activation (Figure 1). Oxidant-induced PKG Iα disulfide bond formation and activation was proposed as a complimentary mechanism to cyclic nucleotide-mediated regulation of kinase activity. Oxidants induce interprotein disulfide bond formation between C42 on each of the adjacent chains in the PKG Iα homodimer complex, rendering the kinase catalytically active independently of cGMP (Burgoyne et al., 2007). Two potential intradisulfide bonds have also been reported to form within PKG Iα in response to metal ion-induced oxidative stress. It was likely that one interdisulfide forms between C117 and C195 and the other between C321 and C516, although it was unclear if both can be present simultaneously within the same monomeric chain Osborne et al., 2011). The redox state of C42 mediating targeting of PKG Iα would appear rationale as it is within the established leucine zipper motif that mediates its interaction with substrates (Michael et al., 2008;Scholten et al., 2008). Perhaps C117-C195 disulfide, which is within a cGMP binding domain and was present within crystallized PKG (Osborne et al., 2011), is a more logical candidate for mediating catalytic competence induced by oxidants. Although it is also notable that C312, which is located within the other cGMP binding pocket, can also form interdisulfide bonds. One possibility is that oxidants induce separate targeting and activating disulfides, with genetic or pharmacological interventions that prevent either of their formation limiting PKG signaling responses to oxidants. Studies with metal ion-induced oxidation identified the intraprotein disulfides, whilst those with H 2 O 2 Osborne et al., 2011), nitrosocysteine (Burgoyne and Eaton, 2009), or H 2 S Osborne et al., 2011) identified the interprotein disulfide. It is possible that each of these oxidants simultaneously induced all of the several disulfides that can form, but they were not reported in some studies because they could not be determined or were not specifically assessed. Conceptually it is possible that there is selectivity in the precise modification different oxidants induce. This is due to different oxidants having distinctive physicochemical properties, together with individual protein thiols having disparate reactivity and solvent accessibility due to their specific environments. Further work would be required to define if a specific ROS species can indeed selectively induce a particular disulfide bond in PKG.
It is evident that PKG Iα can be activated by cGMP binding to the kinase ("classical activation") or alternatively by disulfide bond formation ("oxidant activation"), although the two mechanisms may have some positive cooperativity (Dou et al., 2012). However, cGMP binding to PKG Iα promotes resistance to C42 interprotein disulfide bond formation. Accordingly, cGMP depletion sensitizes PKG Iα to oxidation.

The Role of PKG Iα Disulfide Dimerisation in Blood Vessels
The oxidative activation of PKG Iα by C42 interprotein disulfide formation is an important mechanism contributing to blood pressure homeostasis, being a component of endotheliumderived hyperpolarizing factor (EDHF)-dependent vasodilation (Figure 2). This regulatory mechanism was explored using a knock-in (KI) mouse expressing only a C42S "redox-dead" version of PKG Iα which is unable to form the active disulfide dimer. This subtle, single atom substitution abrogated the vasodilatory action of H 2 O 2 on resistance vessels and resulted in hypertension in vivo (Prysyazhna et al., 2012b). Such oxidative activation of PKG Iα decreases vascular smooth muscle cell Ca 2+, a mechanism that likely contributes to vasodilation induced by oxidants (Muller et al., 2012). PKG Iα C42 interdisulfide activation has been rationalized as an end-effector of EDHF-dependent blood pressure lowering that is mediated by H 2 O 2 derived from uncoupled NOS (Shimokawa, 2014). This mechanism also contributed to human coronary arteriole vasodilation mediated by oxidant-activated PKG opening of smooth muscle voltage and Ca 2+ activated potassium (BK) channels (Zhang et al.,