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Infect. Immun. doi:10.1128/IAI.00527-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The role of Protein kinase A in Trypanosoma cruzi

Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

^* To whom correspondence should be addressed. Email: huangh{at}aecom.yu.edu.

	Abstract

Protein kinase A (PKA) is an important mediator of many signal transduction pathways that occur in eukaryotic cells and it has been implicated as a regulator of stage differentiation in Trypanosoma cruzi. To evaluate the importance of TcPKAc in T. cruzi, a gene encoding a protein kinase A inhibitor (PKI) containing a specific PKA pseudosubstrate, -R-R-N-A-, was subcloned into a pTREX vector and introduced into epimastigotes by electroporation. Expression of PKI has a lethal effect in this parasite. Similarly, a PKA specific inhibitor, H89, killed epimastigotes at the concentration of 10µM. To understand the biology of PKA, the identification of the particular substrates of this enzyme is essential. Using a yeast two-hybrid system, thirty-eight candidates interacting with T. cruzi PKA catalytic subunit (TcPKAc) were identified. Eighteen of these were hypothetical proteins with unknown functions while the others have putative or known functions. The entire open reading frames of eight genes presumably important in regulating T. cruzi growth, adaptation and differentiation, including a type III PI3 kinase (Vps34), a putative PI3 kinase, a putative mitogen-activated protein kinase (ERK), a cAMP-specific phosphodiesterases (PDEC2), a hexokinase, a putative ATPase, a DNA excision repair protein (DERP), and an aquaporin (AQP) were confirmed to interact with TcPKAc in yeast under the highest stringency selection and PKA phosphorylated the recombinant proteins of these genes. Taken together, these findings demonstrate the importance of cAMP-PKA signaling in this organism.