Redox Regulation of cAMP-dependent Protein Kinase Signaling

Abstract

Many components of cellular signaling pathways are sensitive to regulation by oxidation and reduction. Previously, we described the inactivation of cAMP-dependent protein kinase (PKA) by direct oxidation of a reactive cysteine in the activation loop of the kinase. In the present study, we demonstrate that in HeLa cells PKA activity follows a biphasic response to thiol oxidation. Under mild oxidizing conditions, or short exposure to oxidants, forskolin-stimulated PKA activity is enhanced. This enhancement was blocked by sulfhydryl reducing agents, demonstrating a reversible mode of activation. In contrast, forskolin-stimulated PKA activity is inhibited by more severe oxidizing conditions. Mild oxidation enhanced PKA activity stimulated by forskolin, isoproterenol, or the cell-permeable analog, 8-bromo-cAMP. When cells were lysed in the presence of serine/threonine phosphatase inhibitor, NaF, the PKA-enhancing effect of oxidation was blunted. These results suggest oxidation of a PKA-counteracting phosphatase may be inhibited, thus enhancing the apparent kinase activity. Using an in vivo PKA activity reporter, we demonstrated that mild oxidation does indeed prolong the PKA signal induced by isoproterenol by inhibiting counteracting phosphatase activity. The results of this study demonstrate in live cells a unique synergistic mechanism whereby the PKA signaling pathway is enhanced in an apparent biphasic manner. cAMP-dependent protein kinase (PKA or cAPK)2 is a ubiquitously expressed kinase, critical for diverse cellular functions (1). Regulation of the kinase is primarily achieved by the production and degradation of the second messenger cAMP in response to extracellular stimuli, including hormones and neurotransmitters (2). Binding of these ligands to G s -coupled receptors initiates the activation of adenylate cyclase and production of cAMP from ATP. PKA normally exists as an inactive heterotetramer composed of two regulatory subunits and two catalytic subunits (3-5). Binding of cAMP to the regulatory subunits induces the release of the active catalytic subunit, initiating the phosphorylation of numerous downstream substrates, including ion channels at the plasma membrane and sarcoplasmic reticulum (1, 6), the transcription factor cAMPresponse element-binding protein in the nucleus (1, 7-9), and phosphorylase kinase in the cytosol (1, 10). The PKA signal persists until cAMP is hydrolyzed by phosphodiesterases (2) or the free catalytic subunit is inhibited by the heat-stable inhibitor PKI (11). Upon inactivation of PKA by the removal of cAMP, the phosphorylated substrate will persist until reversed by phosphatase activity.Reactive oxygen species are capable of modulating the response of numerous cell signaling pathways (12-15), including that of PKA (16 -21). Oxidants can potentially regulate the PKA pathway at multiple points, including the production and degradation of cAMP, and the phosphorylation and dephosphorylation of substrates. At the level of cAMP production, biochemical evidence has demonstrated that hy...

Cdc42GAP, reactive oxygen species, and the vimentin network

“…Angiotensin II-induced Abl tyrosine phosphorylation is mediated by ROS in arterial smooth muscle cells, which is thought to be critical for signaling induced by the activation of angiotensin subtype 1 receptor (39). In another study, the activity of cAMP-dependent protein kinase (PKA) is regulated by oxidation and reduction (18). In the present study, 5-HT-induced GAP suppression is reversed by the ROS inhibitors.…”

“…The mechanisms by which ROS regulate GAP activity are currently unknown. Since the activity of PKA is directly modulated by redox (18), it is possible that ROS may directly oxidize GAP and thus suppress its activity. Future studies are needed to test the hypothesis.…”

“…ROS have been shown to regulate the activity of protein kinases affecting cellular functions (18,38,39). The role of ROS in regulating GAP activity in mammalian cells in general and in smooth muscle cells in particular is not well understood.…”

Signaling the Signal, Cyclic AMP-dependent Protein Kinase Inhibition by Insulin-formed H2O2 and Reactivation by Thioredoxin

“…In conclusion, this is the first report demonstrating physiologic regulation of type II PKA holoenzyme through its redox state. Finally, redox control of PKA might be shared by alternative physiologic responses as well as by additional key kinases and/or phosphatases (68).…”

Reaktívoxigén-származékok szerepe a szívműködés szabályozásában

“…Ismert, hogy az oxidánsok dózisfüggő bifázisos hatást gyakorolnak a PKA-ra. A tiolcsoportokat oxidáló ágensek alacsony koncentráció mellett fokozzák, míg magas koncentrációban alkalmazva csökkentik a PKAaktivitást [16].…”