Oxidant-induced Activation of Type I Protein Kinase A Is Mediated by RI Subunit Interprotein Disulfide Bond Formation

Abstract

Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for ␣-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of ␤-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase.There is now substantial evidence that oxidant species such as H 2 O 2 are produced in a regulated way in cells where they can function as signaling agents (1, 2). We have been studying the post-translational modification of protein cysteinyl thiols, as this is a major mechanism by which oxidants can alter the structure of proteins and so regulate their function. Our strategy has been to search for proteins that are susceptible to a variety of different modes of cysteine oxidation, such as S-thiolation (3, 4), sulfenation (5), and protein-protein disulfide bond formation (6). The rationale is that once we identify proteins with reactive thiols, the possibility that their oxidation has a functional correlate of physiological significance can be investigated. We previously found the RI regulatory subunits of protein kinase A (PKA) 2 form interprotein disulfide dimers during cardiac oxidative stress (6).Here we investigated the potential impact of this disulfide dimer formation on the function of PKA. PKA has two major forms (type I and type II), both of which exist as a tetramer comprising two catalytic and two regulatory subunits. There are two types of regulatory subunits (RI and RII), the presence of which in the PKA holokinase nominally defines the enzyme as type I or II, respectively. Recent studies have shown that the full dissociation of type I PKA in response to cAMP requires the presence of a substrate (7). This substrateinduced sensitization of type I PKA is not a feature of the type II enzyme (8). The regulatory subunits contain N-terminal sequences that are important for protein kinase A anchor protein (AKAP) binding. AKAPs are a diverse group of proteins that are found next to PKA substrate proteins and, thus, function to target PKA (9). Type I PKA is located in the cytosol, whereas type II is not as a result of being primarily bound (targeted) to AKAP proteins that are associated ...

Deficient angiogenesis in redox-dead Cys17Ser PKARIα knock-in mice

“…1a). Consistent with our past studies 12,13 , we found that H 2 O 2 -or VEGF-induced oxidant formation oxidized PKARIa to a disulphide state (Fig. 1a).…”

“…In this study, we identified PKA as a target of oxidation that can transduce VEGF-dependent oxidant formation into pro-angiogenic signalling. The treatment of BAECs or isolated aortic vessels with VEGF resulted in disulphide dimerization of PKARI associated with its activation 12 . Once activated, oxidized PKA stimulated ERK, a principle mediator of growth factor signalling that couples with upregulation of genes involved in endothelial cell migration and proliferation 21 .…”

“…1a). This disulphide in PKARIa enhances its affinity for A-kinase anchor proteins (AKAPs), serving to target the kinase to substrates it phosphorylates 12,16 . In addition to VEGF, the treatment of BAECs with insulin also induced PKARI oxidation ( Supplementary Fig.…”

“…Although oxidase enzymes mediate angiogenesis, little is known about the molecular targets that sense and transduce the growth-stimulating 'oxidant signal'. Previously we showed the regulatory-RIa subunit of protein kinase A (PKA) can sense oxidants and transduce them into phosphorylation-mediated signalling 12,13 . This is potentially pertinent to angiogenesis as PKA is a widely-appreciated mediator of vessel growth 14,15 .…”

Localization and quaternary structure of the PKA RIβ holoenzyme

“…1B). RIα was shown previously to be targeted only in response to certain stimuli such as capping with an antigen (19) or generation of reactive oxygen species (20). The selective targeting of RIβ seen only when one enriches for mitochondria suggests that RIβ may have a unique role in regulating mitochondrial structure and/or function.…”