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 ...

PKA-dependent, alpha 1-specific NKA activation may be mediated through phosphorylation of the accessory protein PLM, rather than direct alpha1 subunit phosphorylation.

O-Linked N-acetylglucosamine (O-GlcNAc (1-4). In the cell, the HBP converts imported glucose and glucosamine to UDP-GlcNAc. Glutamine:fructose-6-phosphate amidotransferase is the rate-limiting enzyme in this pathway. OGT catalyzes GlcNAc transfer to serine and threonine residues of target proteins, whereas O-O-GlcNAc is known to affect multiple metabolic pathways and has been implicated specifically as a contributor to insulin resistance and type 2 diabetes (5-7). Chronically elevated HBP flux, a result of hyperglycemia, is known to exacerbate metabolic dysregulation in part by targeting metabolic enzymes. For example, in diabetic mice, glycogen synthase (GS) becomes resistant to insulin stimulation as its level of O-GlcNAc modification increases (8, 9). AMP-activated protein kinase is activated in adipocytes with elevated HBP flux, resulting in O-GlcNAc-mediated elevation of fatty acid oxidation (10). To date, the majority of reports of O-GlcNAc-mediated metabolic changes attribute increased O-GlcNAc modification to increased HBP flux. We report a novel and significant induction of O-GlcNAc modification in glucose-deprived HepG2 cells that is independent of increased HBP flux and appears distinct from previously reported stress-induced O-GlcNAc induction. Rather, increased O-GlcNAc with glucose deprivation is mediated by induction of OGT and down-regulation of O-GlcNAcase. Increased O-GlcNAcylation of GS in these conditions contributes to decreased GS activity.