Communication between the regulatory and the catalytic region of the cAMP-responsive guanine nucleotide exchange factor Epac.
Epac1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1 that is directly activated by cAMP. This protein consists of a regulatory region with a cAMP-binding domain and a catalytic region that mediates the GEF activity. Epac is inhibited by an intramolecular interaction between the cAMP-binding domain and the catalytic region in the absence of cAMP. cAMP binding is proposed to induce a conformational change, which allows a LID, an alpha-helix at the C-terminal end of the cAMP-binding site, to cover the cAMP-binding site (Rehmann, H., Prakash, B., Wolf, E., Rueppel, A., de Rooij, J., Bos, J. L., and Wittinghofer, A. (2003) Nat. Struct. Biol. 10, 26-32). Here we show that mutations of conserved residues in the LID region affect cAMP binding only marginally but have a drastic effect on cAMP-induced GEF activity. Surprisingly, some of the mutants have an increased maximal GEF activity compared with wild type. Furthermore, mutation of the conserved VLVLE sequence at the C-terminal end of the LID into five alanine residues makes Epac constitutively active. From these results we conclude that the LID region plays a pivotal role in the communication between the regulatory and catalytic part of Epac.
Rehmann
Holger
H
Department of Physiological Chemistry and Centre of Biomedical Genetics, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
Rueppel
Alma
A
Bos
Johannes L
JL
Wittinghofer
Alfred
A
D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary
D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; D000242:Cyclic AMP