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...
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