Possible roles of protein kinase A in cell motility and excystation of theearly diverging eukaryote Giardia lamblia

Since little is known of how the primitive protozoan parasite, Giardia lamb lia, senses and responds to its changing environment, we characterized a gi ardial protein kinase A (gPKA) catalytic subunit with unusual subcellular l ocalization. Sequence analysis of the 1080-base pair open reading frame sho ws 48% amino acid identity with the cyclic AMP-dependent kinase from Euglen a gracilis, Northern analysis indicated a 1.28-kilobase pair transcript at relatively constant concentrations during growth and encystation, gPKA is a utophosphorylated, although amino acid residues corresponding to Thr-197 an d Ser-338 of human protein kinase A (PKA) that are important for autophosph orylation are absent, Kinetic analysis of the recombinant PKA showed that A TP and magnesium are preferred over GTP and manganese, Kinase activity of t he native PKA has also been detected in crude extracts using kemptide as a substrate. A myristoylated PKA inhibitor, amide 14-22, inhibited excystatio n with an IC50 of 3 muM, suggesting an important role of gPKA during differ entiation from the dormant cyst form into the active trophozoite, gPKA loca lizes independently of cell density to the eight flagellar basal bodies bet ween the two nuclei together with centrin, a basal body/centrosome-specific protein. However, localization of gPKA to marginal plates along the intrac ellular portions of the anterior and caudal pairs of flagella was evident o nly 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.