Simultaneous but Independent Activation of Adenylate Cyclase and Glycosylphosphatidylinositol-Phospholipase C under Stress Conditions in Trypanosoma brucei

doi:10.1074/jbc.271.18.10844

Simultaneous but Independent Activation of Adenylate Cyclase and Glycosylphosphatidylinositol-Phospholipase C under Stress Conditions in Trypanosoma brucei(*)

From the (1)Department of Molecular Biology, University of Brussels, 67 rue des Chevaux, B1640 Rhode St. Genèse, Belgium, the
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom, and the
(2)Department of Biochemistry, Trinity College, University of Dublin, Dublin 2, Ireland

**To whom correspondence should be addressed. Tel.: 32-2-650-96-27; Fax: 32-2-650-96-25.

Abstract

Previous observations suggested a concomitant relationship between the release of the variant surface glycoprotein (VSG) and the activation of adenylate cyclase in the bloodstream form of the parasitic protozoan Trypanosoma brucei. In order to evaluate this hypothesis, adenylate cyclase activity was measured in live trypanosomes subjected to different treatments known to induce the shedding of the VSG coat, namely low pH and trypsin digestion. In both cases adenylate cyclase activation occurred in parallel with the release of the VSG. The latter was found to be mediated by the glycosylphosphatidylinositol-specific phospholipase C that cleaves the glycosylphosphatidylinositol anchor of the protein (VSG lipase). Furthermore, both adenylate cyclase and VSG release were activated by the incubation of trypanosomes with specific inhibitors of protein kinase C, suggesting a repressive role for protein kinase C on both VSG lipase and adenylate cyclase activities. Significantly, in mutant trypanosomes lacking VSG lipase, adenylate cyclase was activated under conditions where VSG release did not occur. Moreover, VSG release was also found to occur in the absence of activation of the cyclase, as observed in the presence of low concentration of the thiol modifying reagent p-chloromercuriphenylsulfonic acid. These observations provide the first demonstration that release of the VSG in response to cellular stress is mediated by the VSG lipase and that while both release of the VSG and activation of adenylate cyclase occur in response to the same stimuli they are not obligatorily coupled.

Footnotes

↵^§ Fellow of the Fonds National de la Recherche Scientifique.
↵^¶ Supported by a fellowship from the European Commission (DGXII).
↵* This work was supported by research contracts with the Communauté Française de Belgique (ARC), by the Belgian Fonds de la Recherche Scientifique (FRSM and Crédit aux Chercheurs), and by the International Brachet Stiftung (IBS).
↵¹ The abbreviations used are:

VSG

variant surface glycoprotein

GTPS

guanosine 5′-O-(thiotriphosphate)

PKA

protein kinase A

PKC

protein kinase C

PMA

phorbol 12-myristate 13-acetate

DMG

1,2-dimyristoyl-rac-glycerol

DAG

diacylglycerol

FCCP

carbonylcyanide p-trifluoromethoxyphenylhydrazone

pCMPS

p-chloromercuriphenylsulfonic acid

TES

2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino} ethanesulfonic acid

MES

4-morpholineethanesulfonic acid

GPI

glycosylphosphatidylinositol

PLC

phospholipase C

PAGE

polyacrylamide gel electrophoresis

CRD

cross-reacting determinant

ESAG

expression site-associated gene.
↵²S. Alexandre, P. Paindavoine, F. Paturiaux-Hanocq, J. Hanocq-Quertier, and E. Pays, unpublished data.
↵³S. Rolin, unpublished data.
↵⁴H. Webb, L. Vanhamme, S. Rolin, D. Le Ray, E. Pays, and M. Carrington, unpublished data.
↵⁵S. Rolin and P. Voorheis, unpublished data.
- Received December 19, 1995.
- Revision received February 16, 1996.