Abstract
Epac is a cAMP-dependent exchange factor for the small GTP-binding protein Rap. The activity of Epac is inhibited by a direct interaction between the C-terminal helical part of the cAMP-binding domain, called the lid, and the catalytic region, which is released after binding of cAMP. Herein, we show that the activation properties are very sensitive to modifications of the cyclic nucleotide. Some analogues are inhibitory and others are stimulatory; some are characterized by a much higher activation potential than normal cAMP. Mutational analysis of Epac allows insights into a network of interactions between the cyclic nucleotides and Epac. Mutations in the lid region are able to amplify or to attenuate selectively the activation potency of cAMP analogues. The properties of cAMP analogues previously used for the activation of the cAMP responsive protein kinase A and of 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclicmonophosphate, an analogue highly selective for activation of Epac were investigated in detail.
Highlights
The exchange protein directly activated by cAMP (Epac)1 is a cAMP-responsive guanine nucleotide exchange factor (GEF) for the small GTP-binding proteins Rap1 and Rap2 [1, 2]
CAMP has the highest affinity (3.3 M) of the cyclic nucleotides tested, the affinities of cIMP (6.4 M), cGMP (14 M), and cXMP (20 M) are of similar magnitude. We investigated whether these nucleotides are able to activate Epac using a fluorescence-based assay, where the ability of Epac to act as a GEF for Rap1 is tested with increasing concentration of nucleotide
To investigate whether the failure of cGMP, cIMP, and cXMP to activate Epac is caused by a failure to bind to full-length Epac149–881, we measured whether these cNMPs were able to inhibit the cAMP-induced
Summary
Exchange protein directly activated by cAMP; GEF, guanine nucleotide exchange factor; DEP, Dishevelled, Egal-10, Pleckstrin; PKA, protein kinase A; wt, wild type; 2Ј-O-Me, 2Ј-O-methyl; 8-pCPT, 8-(4-chlorophenylthio); Rp-cAMPS, adenosine-3Ј,5Ј-cyclicmonophosphorothioate, Rp-isomer; Sp-cAMPS, adenosine-3Ј,5Ј-cyclicmonophosphorothioate, Sp-isomer; PKG, protein kinase G. When analyzing the role of the lid region of Epac in more detail, we observed that a conserved VLVLE sequence in the C-terminal end of the lid region is mediating the inhibition of the catalytic region Mutation of this region resulted in a constitutively active Epac. Single point mutations in the lid region either abolished cAMP-induced activity almost completely or resulted in an activity even higher that of the wt protein. This indicated that after binding of cAMP, the lid region regulates the activation status of Epac [11]. We propose a model of how it communicates with the lid region
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