Possible Roles of Protein Kinase A in Cell Motility and Excystation of the Early Diverging Eukaryote Giardia lamblia

Abstract

Since little is known of how the primitive protozoan parasite, Giardia lamblia, senses and responds to its changing environment, we characterized a giardial protein kinase A (gPKA) catalytic subunit with unusual subcellular localization. Sequence analysis of the 1080-base pair open reading frame shows 48% amino acid identity with the cyclic AMP-dependent kinase from Euglena gracilis. Northern analysis indicated a 1.28-kilobase pair transcript at relatively constant concentrations during growth and encystation. gPKA is autophosphorylated, although amino acid residues corresponding to Thr-197 and Ser-338 of human protein kinase A (PKA) that are important for autophosphorylation are absent. Kinetic analysis of the recombinant PKA showed that ATP and magnesium are preferred over GTP and manganese. Kinase activity of the native PKA has also been detected in crude extracts using kemptide as a substrate. A myristoylated PKA inhibitor, amide 14-22, inhibited excystation with an IC 50 of 3 M, suggesting an important role of gPKA during differentiation from the dormant cyst form into the active trophozoite. gPKA localizes independently of cell density to the eight flagellar basal bodies between the two nuclei together with centrin, a basal body/centrosome-specific protein. However, localization of gPKA to marginal plates along the intracellular portions of the anterior and caudal pairs of flagella was evident only at low cell density and higher endogenous cAMP concentrations or after refeeding with fresh medium. These data suggest an important role of PKA in trophozoite motility during vegetative growth and the cellular activation of excystation.

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]).…”

“…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]). Activity of cell signaling proteins is often regulated by phosphorylation and dephosphorylation, leading to complex signaling networks.…”

“…Cytoskeletons were extracted in situ for 10 min with 0.5% Triton X-100 in 10 mM Tris (pH 8.3), 2 mM EDTA, 2 mM MgCl2, 150 mM KCl, and 2 mM dithiotreitol [24]. Whole trophozoites were fixed in methanol and permeabilized for 10 min with 0.5% Triton X-100 in PBS [7]. Water-treated and excysting cysts (in Stages 1 and 2 and 30 and 60 min excystation) were dried on the coverslips.…”

“…P30153), respectively. The major difference is that the gPP2A A subunits have divergent amino acid inserts (50AA between AA 283 and 341 in ORF 7439 and several small inserts in ORF 16567 among a 19AA one between AA 275 and AA 293), which are common amongst conserved giardial genes [7,35]. There are several PP2A regulatory B subunits and blast searches identified 8 putative B subunits in the Giardia genome (Table 1).…”

“…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.…”