Epac1 and cAMP-dependent Protein Kinase Holoenzyme Have Similar cAMP Affinity, but Their cAMP Domains Have Distinct Structural Features and Cyclic Nucleotide Recognition

Abstract

The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a K d value (2.9 M) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had K d values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RI␣ led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.Lower eukaryotes like Saccharomyces cerevisiae have as sole receptor for the signaling molecule cAMP the two cAMP-binding sites (A and B) of the regulatory (R) 4 subunit of the cAMPdependent protein kinase (PKA). These tandem cAMP binding domains can be traced in all four isoforms (RI␣, RI␤, RII␣, and RII␤) of mammalian PKA (1), in the cGMP-dependent protein kinases (2, 3), the cyclic nucleotide gated ion channels (3-5), and the exchange proteins directly activated by cAMP, Epac1, and Epac2 (6). In PKA conformational changes induced by cAMP binding to both site A and B are required to dissociate the catalytic (C) subunit from the holoenzyme complex (7,8).In contrast, cAMP binding to a single site of Epac is sufficient to relieve the tonic intrachain inhibition of its GDP exchange activity toward the small GTPase Rap (6, 9). A major issue in cell signaling is how the second messenger cAMP uses the receptors PKA and Epac to coordinate biological effects (10). Comparison of the cAMP affinity of Epac1 and PKA holoenzyme would help predict which of the two cAMP receptors, if present in the same compartment, is likely to be preferentially activated by a slight increase of cAMP. For this the cAMP affinity of PKA holoenzyme, so far unknown, must be determined. The functional cAMP site in Epac is presumably derived from the B site of PKA b...

The cyclic AMP effector Epac integrates pro- and anti-fibrotic signals

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Identification and Validation of Modulators of Exchange Protein Activated by cAMP (Epac) Activity

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