Abstract
Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H(2)O(2) generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H(2)O(2) added before cAMP suppressed cAMP activation of type IIbeta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H(2)O(2) impaired activation of type IIalpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIalpha and catalytic alpha subunits. H(2)O(2) was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic alpha subunit, which is active independently of cAMP activation, and (c) if the catalytic alpha subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H(2)O(2) inhibition of PKA activation remained after H(2)O(2) elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H(2)O(2) incubation. These data strongly suggest that H(2)O(2) promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIalpha or IIbeta subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic alpha subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.
Highlights
protein kinase (PKA) Regulation by H2O2 administration has been reported [7]; chronic hyperinsulinemia resulted in enhanced cAMP production through -adrenergic receptor activation in adipocytes [8]
Insulin in combination with Mn2ϩ or GTP␥S promoted H2O2 production from NADPH, (Fig. 1b), similar to the manner in which this occurs in plasma membranes of human adipocytes incubated with GTP␥S, NADPH, and insulin [31] and in 3T3-L1 adipocytes challenged with insulin [11, 50]
Experiments to analyze the role of Epac in adipose cell lipolysis response were conducted, because previous reports showed the participation of Epac in two cases of cross-talk between insulin and cAMP signaling pathways [6, 7]
Summary
PKA Regulation by H2O2 administration has been reported [7]; chronic hyperinsulinemia resulted in enhanced cAMP production through -adrenergic receptor activation in adipocytes [8]. Oxidative stress can lead to the formation of a large number of protein disulfides [17,18,19], and the cytosol is a reducing environment, the thiol status of proteins may be more dynamic than appreciated previously [20] It has been recently demonstrated in isolated rat ventricular myocytes that type I PKA is redox-active, forming an interprotein disulfide bond between two cysteines that align antiparallel to each other on different type I regulatory subunits (RIS) in response to cellular H2O2 [21]. By use of a fully active H2O2-insensitive C199A catalytic subunit [26] and supported by phylogenetic analysis of protein kinases, the highlighted Cys residues in PKA holoenzyme that are oxidized by H2O2, preventing cAMP physiologic activation, were identified
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