Potential Involvement of Extracellular Signal-regulated Kinase 1 and 2 in Encystation of a Primitive Eukaryote, Giardia lamblia

Abstract

Mitogen-activated protein kinase (MAPK) pathways are major signaling systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified Giardia lamblia homologues of two members of the MAPK family ERK1 and ERK2. Functional characterization of giardial ERK1 and ERK2 revealed that both kinases were expressed in trophozoites and encysting cells as 44-and 41-kDa polypeptides, respectively, and were catalytically active. Analysis of the kinetic parameters of the recombinant proteins showed that ERK2 is ϳ5 times more efficient than ERK1 in phosphorylating myelin basic protein as a substrate, although the phosphorylating efficiency of the native ERK1 and ERK2 appeared to be the same. Immunofluorescence analysis of the subcellular localization of ERK1 and ERK2 in trophozoites showed ERK1 staining mostly in the median body and in the outer edges of the adhesive disc and ERK2 staining in the nuclei and in the caudal flagella. Our study also showed a noticeable change in the subcellular distribution of ERK2 during encystation, which became more punctate and mostly cytoplasmic, but no significant change in the ERK1 localization at any time during encystation. Interestingly, both ERK1 and ERK2 enzymes exhibited a significantly reduced kinase activity during encystation reaching a minimum at 24 h, except for an initial ϳ2.5-fold increase in the ERK1 activity at 2 h, which resumed back to the normal levels at 48 h despite no apparent change in the expression level of either one of these kinases in encysting cells. A reduced concentration of the phosphorylated ERK1 and ERK2 was also evident in these cells at 24 h. Our study suggests a functional distinction between ERK1 and ERK2 and that these kinases may play a critical role in trophozoite differentiation into cysts.Giardia lamblia, an evolutionary primitive eukaryotic protozoan parasite and an intestinal pathogen of humans and animals, is one of the major causes of water-borne diseases worldwide (1). This flagellated protozoan undergoes complex life cycle stages while inside the host. Exposure to the highly acidic condition in stomach and proteases in the upper small intestine triggers excystation of trophozoites from the ingested cysts. Newly emerged trophozoites swim freely in the intestinal fluid and colonize the upper small intestine to replicate (2, 3). As enterocytes migrate to the tip of the villus and get sloughed off into the intestinal lumen, the attached trophozoites either reattach to new enterocytes to remain in the intestine or differentiate into infective cysts. Although the life cycle of this primitive eukaryote and physiological signals that regulate induction of excystation and encystation have been studied extensively, the molecular mechanisms by which trophozoites sense and respond quickly to the environmental signals in the intestine to initiate encystation remain largely unknown.Encystation is an adaptive process to cope with the depletion of nutrients, specifically cholester...

Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation

“…In Giardia trophozoites, gPP2A-C localizes to the eight basal bodies, as previously reported for giardial PKAc/r, the MAP kinase ERK1, centrin, and calmodulin [7][8][9]24,38,39]. gPP2A-C also localizes to the ventral disk (similar to ERK 1 [9]), to the anterior and caudal PDR (similar to PKAc/r [7,8]) and to the posterior-lateral PDR (similar to centrin [24]).…”

“…Nonetheless, the signal transduction pathways leading to the cytoskeletal changes during differentiation are largely unknown. Previously, it was shown that protein kinase A (PKA) and calmodulin both localize to the Giardia cytoskeleton and regulate different steps in the resumption of motility and cytokinesis during the cellular awakening of excystation [7], while PKA and ERK kinases have been implicated in encystation [8,9]. These data suggest that specific cell signaling mechanisms may regulate cytoskeletal functions during differentiation.…”

Encystation commitment inGiardia duodenalis: a long and winding road

“…Parasite, 2009, 16, 247-258 Transcription factor mSREBP translocates to the nucleus where it interacts with genomic SRE at gene promoters of either the cholesterol biosynthesis pathway (HMGCoAsyn and HMGCoA-red) and fatty acid biosynthesis (LDLr) and CWPs resulting in their up-regulation (Worgall et al, 2004;Ellis et al, 2003) (Fig. 3).…”

“…The transcriptional activity of mSREBP is decreased by its ubiquitylation and/or sumoylation while the effect of its phosphorylation depends on the kinase involved: extracellular-signal regulated kinase (ERK)-mediated phosphorylation increases transcription by mSREBP while it decreases by PKA or glycogen synthase kinase (GSK)-mediated phosphorylation (Arito et al, 2008). Giardial homologues of the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 have been identified (Ellis et al, 2003). Endogenous ERK1 exhibited increased activity, slightly higher phosphorylation and a noticeable partial relocalization to the cell membrane at an early time post-encystation induction (≈ 2 hrs; see Figs.…”

“…Endogenous ERK1 exhibited increased activity, slightly higher phosphorylation and a noticeable partial relocalization to the cell membrane at an early time post-encystation induction (≈ 2 hrs; see Figs. 6A and 6B of Ellis et al, 2003) while ERK2 at this stage relocates from the membrane to the cytosol concomitant with a decrease in its activity (Ellis et al, 2003). Interestingly, Giardia and Dictyostelium discoideum ERK2s are closely related at the amino acid (aa) level (66 % identity and 84 % homology, expectation value of 8e-130) (Table I) with a co-localized cAMPdependent PKA phosphorylation site (KRiT) that in the slime mold is involved in activation and translocation of ERK2 to the nucleus during the multicellular development response to extracellular factors that increase intracellular cAMP levels.…”