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

Erika S Abel,Barbara J Davids,Liza D Robles,Clarence E Loflin,Frances D Gillin,Ratna Chakrabarti

doi:10.1074/jbc.m006589200

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 microm, 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.

Highlights

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 catalytic subunit with unusual subcellular localization
Sequence and Expression Analyses of giardial protein kinase A (gPKA)—Upon screening a Giardia cDNA library with a 250-base pair probe derived from conserved protein kinase A (PKA) sequences, a 1.28-kb fragment containing an ORF of 1.08 kb (360 amino acids) corresponding to a Giardia homologue of PKA catalytic subunit was cloned (Fig. 1)
Since the serine-threonine kinase family exhibits a significant degree of conservation of the kinase domains, the gPKA ORF showed limited homology with a number of other serine threonine kinases, such as cGMPdependent protein kinases

Summary

Introduction

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. A myristoylated PKA inhibitor, amide 14-22, inhibited excystation with an IC50 of 3 ␮M, suggesting an important role of gPKA during differentiation from the dormant cyst form into the active trophozoite. 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.

Results

Conclusion