Molecular cloning and characterization of mitogen-activated protein kinase 2 in Trypanosoma cruzi

Abstract

Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified a Trypanosoma cruzi homologue of the MAPK family that we have called TcMAPK2. Sequence analyses demonstrates TcMAPK2 has high homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. Enzymatic assays of both recombinant TcMAPK2 and native protein obtained by immunoprecipitation using anti-TcMAPK2 demonstrated that both preparations of TcMAPK2 were catalytically active. Immunofluorescence analysis of the subcellular localization of TcMAPK2 determined it is mainly cytoplasmic in epimastigotes, along the flagella in trypomastigotes and on the plasma membrane of intracellular amastigotes. Phosphorylated TcMAPK2 was highest in trypomastigotes and lowest in amastigotes. Recombinant TcMAPK2 was able to phosphorylate the recombinant protein of a cAMP specific phosphodiesterase. Overexpression of TcMAPK2 in epimastigotes inhibited growth and development leading to death. TcMAPK2 has an important role in the stress response of the parasite and may be important in regulating proliferation and differentiation.

Figure 1

Comparison of the TcMAPK2 with other MAPKs. Amino acid sequence alignment of TcMAPK2 from different species. TcMAPK2 (TcERK₂) in the T. cruzi genome (Tc00.1047053510295.50 and Tc00.1047053506007.40); GIERK2: Giardia intestinalis ERK2 (GenBank accession: AAN73430.1). Lm-MAPK2: Leishmania major (MAPK Gene ID: 5655322 LmjF36.072); HuMAPK15: a human MAPK 15 (DBSOURCE: accession CH471162.2). TcMAPK2 includes consensus in subdomain I, GXGXXGXV (GQGAYGIV 20–27); II, AXK (ALK 40–42); III, RXXRE (RTFRE 56–60); VI, HRDXKPXN (HRDMKPSN, 133–140); VII, DFG (DFG, 153–155); VIII, TXYXXXRXYRXPE (TDYIMTRWYRPPE, 174–186). Typical features of MAPK exist in this kinase. Subdomains I–III are for binding and orienting ATP. The conserved TXY motif for dual phosphorylation (Thr¹⁷⁴–Tyr¹⁷⁶) for the activation of the enzyme is also present. The shaded residues are those that are found in the consensus sequence.

Figure 2

Expression and purification of TcMAPK2 and analyses of recombinant and nativeTcMAPK2. (A) Coomassie blue R250 staining lane 1: 20 µl of non-induced bacteria; lane 2: 20 µl of 1 mM IPTG IP induced bacteria; lane 3: first eluate; lane 4: second eluate; lane 5: third eluate from the Trap FF crude column. (B) Immunoblot using HIS₆ mAb. Immunoblot examined identical samples in a duplicate SDS gel using antibody to HIS₆. Note that the expression and purification technique for TcMAPK2 was successful (see Materials and Methods for details). (C) After in vitro phosphorylation reaction, the samples were subjected to the resolution of SDS gel and Coomassie blue R250 staining. The purified recombinant TcMAPK2 and the substrates were visualized in the gel. MBP concentrations were indicated at the bottom of each lane. (D) The same gel was dried and used for autoradiography for five minutes. Phosphorylation of MBP and autophosphorylation of TcMAPK2 are detected by autoradiography. (E) Anti-TcMAPK2 precipitated immune complexes from epimastigotes phosphorylate MBP. The bead with immune complexes was resuspended in 50 µl of buffer A and then used for kinase assay using MBP as a substrate. “Neg” indicates the negative control (using 16 µl bead suspension from parasite lysate with pre-immune serum). 4 µl, 8 µl and 16 µl indicate the amount of bead suspension with Anti-TcMAPK2 immune complexes. Note that Anti-TcMAPK2 immune complexes can phosphorylate MBP in the kinase assay, indicating that native TcMAPK2 is active.

Figure 3

Biochemical analysis of TcMAPK2. (A) Computer software analysis of enzymatic activity of TcMAPK2. Enzymatic parameters were calculated using KaleidaGraph 4 software. At the standard reaction conditions using 5 µg of the recombinant enzyme and varying concentration of MBP, the specific activity of recombinant TcMAPK2 is 6.4 ± 0.5 nmole PO₄/incorp./µmole TcMAPK2/second and the apparent K_m is in the nanomolar range (1.2 ± 0.2 nM). (B) EDTA abolished the enzymatic activity. Light color columns are reactions without EDTA while dark color columns with 250 mM EDTA. Three separate replicates were performed. (C) TcMAPK2 is Mg²⁺ dependent. 10 mM of either Ca²⁺ or Mn²⁺ shows no effect on the enzymatic activity as compared to standard kinase assay without adding divalent metal compounds while 10 mM Mg²⁺ significantly increases more than three folds of the enzymatic activity. The plot is a representative of three separate experiments. (D) TcMAPK2 is ATP dependent. Increase the concentration of ATP increases the incorporation of phosphate into the MBP. This linear curve is a representative of three separate experiments.

Figure 4

Developmental studies of TcMAPK2 in three forms of T. cruzi. (A) Northern blot analysis of T. cruzi. Try (trypomastigotes), Epi (epimastigotes) and Ama (extracellular amastigotes). Top panel shows TcMAPK2 probe hybridization signals (∼4 kb) of total RNAs from three life forms of T. cruzi. Bottom panel shows Ethidium bromide-stained gels demonstrated the equal loading of T. cruzi ribosomal RNA and confirmed the integrity of the samples. There was no change in the level of mRNA among three forms of T. cruzi. (B) Immunoblot analysis of T. cruzi using anti-TcMAPK2. The results indicated that the TcMAPK2 protein level was at the same level in trypomastigotes and epimastigotes but TcMAPK2 protein level in amastigotes is lower than that in other forms. (C) Immunoblot analysis of T. cruzi using anti-phospho-TcMAPK2. Trypomastigotes have highest level of phospho-TcMAPK2, followed by epimastigote and then extracellular amastigotes. Based on the data from analysis of densitometry, the ratio of phospho-TcMAPK2 vs. TcMAPK2 is two folds higher in trypomastigotes as compared to that of epimastigotes and amastigotes.

Figure 5

IFA of T. cruzi with anti-TcMAPK2. Epi indicates epimastigotes; Try indicates trypomastigotes, Intra Ama indicates intracellular amastigotes and Ama indicates amastigotes (newly released). IFA revealed cytoplasmic staining in epimastigotes. In addition, there are some intensive spots staining in the flagellar side (top part). Staining in trypomastigotes was evident along the flagellar side (second part). Staining of intracellular amastigotes or newly released amastigotes shows that TcMAPK2 mainly localized in the plasma membrane (third and bottom parts). Pre-immure sera did not show any staining (not shown). Scale bars = 10 µm.

Figure 6

TcMAPK2 phosphorylates TcPDEC2. Top part, Coomassie blue R250 staining; Bottom part, the same gel was used for autoradiography. Lane1, purified recombinant TcPDEC2 alone (without purified recombinant TcMAPK2). Lane2 purified recombinant TcMAPK2 and purified recombinant TcPEDC2 together for in vitro kinase assay reaction (see Materials and Methods for details). Note that TcMAPK2 phosphorylated TcPEDC2. Also, autophosphorylation occurred in TcMAPK2.