A Novel Adenylyl Cyclase Detected in Rapidly Developing Mutants of Dictyostelium

doi:10.1074/jbc.273.47.30859

A Novel Adenylyl Cyclase Detected in Rapidly Developing Mutants of *Dictyostelium**

From the^‡Department of Biochemistry, University of Oxford, Oxford OX13QU, United Kingdom and the^§Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, 2333 AL Leiden, The Netherlands

Abstract

Disruption of either the RDEA orREGA genes leads to rapid development inDictyostelium. The RDEA gene product displays homology to certain H2-type phosphotransferases, while REGAencodes a cAMP phosphodiesterase with an associated response regulator. It has been proposed that RDEA activates REGA in a multistep phosphorelay. To test this proposal, we examined cAMP accumulation inrdeA and regA null mutants and found that these mutants show a pronounced accumulation of cAMP at the vegetative stage that is not observed in wild-type cells. This accumulation was due to a novel adenylyl cyclase and not to the known Dictyosteliumadenylyl cyclases, aggregation stage adenylyl cyclase (ACA) or germination stage adenylyl cyclase (ACG), since it occurred in anacaA/rdeA double mutant and, unlike ACG, was inhibited by high osmolarity. The novel adenylyl cyclase was not regulated by G-proteins and was relatively insensitive to stimulation by Mn²⁺ ions. Addition of the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) permitted detection of the novel adenylyl cyclase activity in lysates of anacaA/acgA double mutant. The fact that disruption of theRDEA gene as well as inhibition of the REGA-phosphodiesterase by IBMX permitted detection of the novel AC activity supports the hypothesis that RDEA activates REGA.

Footnotes

↵* This work was supported by Biotechnology and Biological Sciences Research Council Grant ICR 07521.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed (P. Schaap or J. D. Gross): Cell Biology Section, Institute for Molecular Plant Sciences, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. Tel.: 31-71-5274927; Fax: 31-71-5274999; E-mail: schaap{at}rulbim.leidenuniv.nl(for P. Schaap) or Dept. of Biochemistry, University of Oxford, Oxford OX13QU, United Kingdom. Fax: 44-1865-275259; E-mail:gross{at}bioch.ox.ac.uk (for J. D. Gross).
↵2 M. Meima and P. Schaap, unpublished results.
Abbreviations:

PKA

cAMP-dependent protein kinase

PKA-R

PKA regulatory subunit

ACA

aggregation stage adenylyl cyclase

ACG

germination stage adenylyl cyclase

IBMX

3-isobutyl-1-methylxanthine

PDE

phosphodiesterase

2′H-cAMP

2′-deoxyadenosine 3′,5′-monophosphate

GDPβS

guanosine 5′-O-(2-thiodiphosphate)

GTPγS

guanosine 5′-O-(3-thiotriphosphate)

DTT

dithiothreitol

PB

phosphate buffer

ACB

adenylyl cyclase B.
- Received August 20, 1998.
- Revision received September 24, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.