Signaling the signal, cyclic AMP-dependent protein kinase inhibition by insulin-formed H2O2 and reactivation by thioredoxin

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H2O2 generated by insulin in rat adipocytes impaired cAMP- mediated amplification cascade of lipolysis. These micromolar concentrations of H2O2 added before cAMP suppressed cAMP activation of type II beta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H2O2 impaired activation of type II alpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory II alpha and catalytic alpha subunits. H2O2 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. H2O2 inhibition of PKA activation remained after H2O2 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 H2O2 incubation. These data strongly suggest that H2O2 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 II alpha or II beta 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.