Ligand-mediated Activation of the cAMP-responsive Guanine Nucleotide Exchange Factor Epac*

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.

[1]  S. Døskeland,et al.  cAMP Analog Mapping of Epac1 and cAMP Kinase , 2003, Journal of Biological Chemistry.

[2]  A. Wittinghofer,et al.  Communication between the Regulatory and the Catalytic Region of the cAMP-responsive Guanine Nucleotide Exchange Factor Epac* , 2003, Journal of Biological Chemistry.

[3]  Andre Hoelz,et al.  Structural Evidence for Feedback Activation by Ras·GTP of the Ras-Specific Nucleotide Exchange Factor SOS , 2003, Cell.

[4]  J. Bos,et al.  Epac-selective cAMP Analog 8-pCPT-2′-O-Me-cAMP as a Stimulus for Ca2+-induced Ca2+ Release and Exocytosis in Pancreatic β-Cells* , 2003, The Journal of Biological Chemistry.

[5]  S. Døskeland,et al.  A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK , 2002, Nature Cell Biology.

[6]  A. Wittinghofer,et al.  Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1. , 2001, Journal of molecular biology.

[7]  A. Wittinghofer,et al.  Mechanism of Regulation of the Epac Family of cAMP-dependent RapGEFs* , 2000, The Journal of Biological Chemistry.

[8]  A M Graybiel,et al.  A family of cAMP-binding proteins that directly activate Rap1. , 1998, Science.

[9]  A. Wittinghofer,et al.  Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP , 1998, Nature.

[10]  S. Taylor,et al.  Dissecting cAMP binding domain A in the RIalpha subunit of cAMP-dependent protein kinase. Distinct subsites for recognition of cAMP and the catalytic subunit. , 1998, The Journal of biological chemistry.

[11]  John Kuriyan,et al.  The structural basis of the activation of Ras by Sos , 1998, Nature.

[12]  A. Wittinghofer,et al.  Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm. , 1998, Biochemistry.

[13]  A. Wittinghofer,et al.  Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N) , 1997, Oncogene.

[14]  S. Lohmann,et al.  Fast and Slow Cyclic Nucleotide-dissociation Sites in cAMP-dependent Protein Kinase Are Transposed in Type Iβ cGMP-dependent Protein Kinase* , 1996, The Journal of Biological Chemistry.

[15]  N. Xuong,et al.  Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains , 1995, Science.

[16]  L. Alberghina,et al.  The minimal active domain of the mouse ras exchange factor CDC25Mm. , 1995, Biochemical and biophysical research communications.

[17]  J. Corbin,et al.  Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity. , 1991, The Journal of biological chemistry.

[18]  J. Corbin,et al.  One amino acid change produces a high affinity cGMP-binding site in cAMP-dependent protein kinase. , 1990, The Journal of biological chemistry.

[19]  B. Jastorff,et al.  Inhibition of cGMP‐dependent protein kinase by (Rp)‐guanosine 3',5'‐monophosphorothioates , 1990, FEBS letters.

[20]  I. Weber,et al.  Predicted structures of the cGMP binding domains of the cGMP-dependent protein kinase: a key alanine/threonine difference in evolutionary divergence of cAMP and cGMP binding sites. , 1989, Biochemistry.

[21]  D. Øgreid,et al.  Studies of cGMP analog specificity and function of the two intrasubunit binding sites of cGMP-dependent protein kinase. , 1986, The Journal of biological chemistry.

[22]  W. Stec,et al.  Competitive cAMP antagonists for cAMP-receptor proteins. , 1984, The Journal of biological chemistry.

[23]  S. Døskeland,et al.  Evidence that cyclic nucleotides activating rabbit muscle protein kinase I interact with both types of cAMP binding sites associated with the enzyme. , 1983, The Journal of biological chemistry.

[24]  J. Miller,et al.  Mapping adenosine cyclic 3',5'-phosphate binding sites on type I and type II adenosine cyclic 3',5'-phosphate dependent protein kinases using ribose ring and cyclic phosphate ring analogues of adenosine cyclic 3',5'-phosphate. , 1981, Biochemistry.

[25]  A. Wittinghofer,et al.  Structure and regulation of the cAMP-binding domains of Epac2 , 2003, Nature Structural Biology.

[26]  N. Xuong,et al.  Molecular basis for regulatory subunit diversity in cAMP-dependent protein kinase: crystal structure of the type II beta regulatory subunit. , 2001, Structure.

[27]  B. Jastorff,et al.  [15] cAMP analog antagonists of cAMP action , 1988 .