Possible roles of protein kinase A in cell motility and excystation of the early diverging eukaryote Giardia lamblia.
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.
Abel
E S
ES
Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, Florida 32826, USA.
Davids
B J
BJ
Robles
L D
LD
Loflin
C E
CE
Gillin
F D
FD
Chakrabarti
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002384:Catalysis; D002454:Cell Differentiation; D018385:Centrosome / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D018076:DNA, Complementary / Q000378:metabolism; D005407:Flagella / Q000378:metabolism; D017353:Gene Deletion; D015723:Gene Library; D016829:Giardia lamblia / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009068:Movement / Q000502:physiology*; D009227:Myristic Acids / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D016366:Open Reading Frames; D010766:Phosphorylation; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic
D000596:Amino Acids; D018076:DNA, Complementary; D009227:Myristic Acids; D009842:Oligopeptides; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; C037642:kemptide; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases