Biochem. J. (2007) 401
(309–316) (Printed in Great Britain)
Pharmacological profiling of the Dictyostelium adenylate cyclases ACA, ACB and ACG
Elisa ALVAREZ-CURTO, Karin E. WEENING and Pauline SCHAAP1
School of Life Sciences, University of Dundee, Dundee, Scotland, U.K.
Intracellular and secreted cAMPs play crucial roles in controlling cell movement and gene regulation throughout development of the social amoeba Dictyostelium discoideum. cAMP is produced by three structurally distinct ACs (adenylate cyclases), ACA, ACG and ACB, which have distinctive but overlapping patterns of expression and, as concluded from gene disruption studies, seemingly overlapping functions. In addition to gene disruption, acute pharmacological abrogation of protein activity can be a powerful tool to identify the protein's role in the biology of the organism. We analysed the effects of a range of compounds on the activity of ACA, ACB and ACG to identify enzyme-specific modulators. Caffeine, which was previously used to specifically block ACA function, also inhibited cAMP accumulation by ACB and ACG. IPA (2´,3´-O-isopropylidene adenosine) specifically inhibits ACA when measured in intact cells, without affecting ACB or ACG. All three enzymes are inhibited by the P-site inhibitor DDA (2´,5´-dideoxyadenosine) when assayed in cell lysates, but not in intact cells. Tyrphostin A25 [a-cyano-(3,4,5-trihydroxy)cinnamonitrile] and SQ22536 [9-(tetrahydro-2´-furyl)adenine] proved to be effective and specific inhibitors for ACG and ACA respectively. Both compounds acted directly on enzyme activity assayed in cell lysates, but only SQ22536 was also a specific inhibitor when added to intact cells.
Key words: adenylate cyclase, caffeine, cAMP, enzyme-specific inhibitor, P-site inhibition, Dictyostelium discoideum.
Abbreviations used: AC, adenylate cyclase; cAR1, cAMP receptor 1; DA, 2´-deoxyadenosine; DcAMP, 2´-deoxyadenosine 3´,5´-monophosphate; DDA, 2´,5´-dideoxyadenosine; DTT, dithiothreitol; GTP[S], guanosine 5´-[g-thio]triphosphate; IBMX, isobutylmethylxanthine; IPA, 2´,3´-O-isopropylidene adenosine; PDE, phosphodiesterase; PdeE, phosphodiesterase E; PdsA, phosphodiesterase A; PKA-R, protein kinase A regulatory subunit; RdeA, phospho-relay intermediate A; RegA, phosphodiesterase 2.
1To whom correspondence should be addressed, at MSI/WTB/JBC Complex, Dow Street, Dundee DD1 5EH, Scotland, U.K. (email p.schaap@dundee.ac.uk).
Received 13 June 2006/22 August 2006; accepted 5 September 2006
Published as BJ Immediate Publication 5 September 2006, DOI 10.1042/BJ20060880
The Biochemical Society, London ©2007
Editorial Advisory Panel
RSS feeds
Table of contents by email
My BJ toolbox
