Role of protein kinase A in Trypanosoma cruzi

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 the PKA catalytic subunit of T. cruzi (TcPKAc), a gene encoding a PKA 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 pharmacological inhibitor, H89, killed epimastigotes at a concentration of 10 muM. To understand the biology of PKA, identification of the particular substrates of this enzyme is essential. Using a yeast two-hybrid system, 38 candidates interacting with TcPKAc were identified. Eighteen of these were hypothetical proteins with unknown functions, while the others had 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 extracellular signal-regulated kinase, a cyclic AMP (cAMP)-specific phosphodiesterase (PDEC2), a hexokinase, a putative ATPase, a DNA excision repair protein, and an aquaporin were confirmed to interact with TcPKAc in the yeast Saccharomyces cerevisiae under the highest stringency selection conditions, and PKA phosphorylated the recombinant proteins of these genes. Taken together, these findings demonstrate the importance of cAMP-PKA signaling in this organism.

FIG. 1.

Eight candidates with defined or presumably important functions interact with TcPKAc. Using a yeast two-hybrid system, eight genes encoding important proteins in target plasmids interacting with pBD-TcPKAc were obtained. All ORFs of these genes were subcloned into a pAD-Gal4 vector. Each pAD-Gal4 construct was cotransformed into yeast with pBD-TcPKAc and cultured under high-stringency conditions (-Leu, -Trp, -His). These genes are shown on a composite plate, as follows: 1, AQP 9 (TcAQP) (Tc00.1047053508257.140); 2, ATPase (Tc00.1047053508903.100); 3, DERP (Tc00.1047053506983.60); 4, PK (putative ERK homolog) (Tc00.1047053510295.50); 5, hexokinase (Tc00.1047053508951.20); 6, cAMP PDEC2 (GenBank accession no. DQ008164); 7, phosphatidylinositol 3-kinase 2 (PI3 kinase) (Tc00.1047053508859.90); 8, class III phosphatidylinositol 3-phosphate kinase (VPs34) (Tc00.1047053511903.160); 9, positive control (pADwt with pBDwt); and 10, negative control (pADwt with pLaminC).

FIG. 2.

ERK interacted with PDEC2. Cotransformation of pAD-Gal4-PDEC2 with pBD-Gal4-ERK was performed in yeast under the highest stringency conditions. This composite plate demonstrated that these two candidates interacted in this system. 1, pAD-Gal4-PDEC2 with pBD-Gal4-ERK; 2, positive control (pADwt with pBDwt); and 3, negative control (pADwt with pLaminC).

FIG. 3.

TcPKAc MAb interacted with TcAQP in vivo. Coimmunoprecipitation demonstrated that TcPKAc MAb precipitated a complex containing a 26-kDa band which reacted with the TcAQP antibody, indicating that two proteins, TcPKAc and TcAQP, interacted in this organism. 1, negative control using an unrelated MAb (anti-BAG5 of Toxoplasma gondii); 2 and 3, TcPKAc MAb (for details, see Materials and Methods).

FIG. 4.

PKAc phosphorylates important proteins in T. cruzi. In vitro phosphorylation of recombinant proteins important in T cruzi by PKA is confirmed. This composite figure demonstrated that eight proteins were phosphorylated by PKA in vitro. Lane 1, absence of recombinant protein as negative control; lane 2, various recombinant proteins were phosphorylated in the absence of PKI; and lane 3, PKI inhibited the phosphorylation. −, absence of reagent; +, presence of reagent. Arrowheads indicate each of the recombinant proteins and their molecular mass. PI3, phosphatidylinositol 3-kinase 2 (PI3 kinase) (Tc00.1047053508859.90); VPS 34, class III phosphatidylinositol 3-phosphate kinase (VPs34) (Tc00.1047053511903.160); PDEC2, cAMP PDEC2 (GenBank accession no. DQ008164); DERP (Tc00.1047053506983.60); HEXO, hexokinase (Tc00.1047053508951.20); ERK, putative ERK homolog (Tc00.1047053510295.50); ATPase, putative ATPase (Tc00.1047053508903.100); and AQP, AQP 9 (TcAQP) (Tc00.1047053508257.140).