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J. Biol. Chem., Vol. 283, Issue 18, 12373-12386, May 2, 2008

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

Martha Zentella de Piña, Héctor Vázquez-Meza, Juan Pablo Pardo, Juan Luis Rendón, Rafael Villalobos-Molina, Héctor Riveros-Rosas, and Enrique Piña¹

From the Departamento de Bioquímica, Facultad de Medicina, and Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, México, DF 04510, México

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 IIβ 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 PKA holoenzyme from bovine heart and from that reconstituted with regulatory II and catalytic subunits. H₂O₂ was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic subunit, which is active independently of cAMP activation, and (c) if the catalytic 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 or IIβ subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.

Received for publication, August 16, 2007 , and in revised form, February 1, 2008.

^* This work was supported by Consejo Nacional de Ciencia y Tecnología, México, Grant 45003M (to E. P.) and by Dirección General de Asuntos del Personal Académico, National Autonomous University of Mexico, Grants IN202106-2 and IN224206 (to E. P. and H. R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Appendix 1.

¹ To whom correspondence should be addressed: Dept. Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado Postal 70159, México DF 04510, México. Tel.: 52-55-5622-0829; Fax: 52-55-5616-2419; E-mail: epgarza{at}servidor.unam.mx.

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