Molecular cloning and expression of the catalytic subunit of protein kinase A from Trypanosoma cruzi.
The activation of protein kinase A (cyclic adenosine monophosphate-dependent protein kinase) by cyclic adenosine monophosphate is believed to play an important role in regulating the growth and differentiation of Trypanosoma cruzi. A PCR using degenerate oligonucleotide primers against conserved motifs in the VIb and VIII subdomains of the ACG family of serine/threonine protein kinases was utilised to amplify regions corresponding to the parasite homologue of the protein kinase A catalytic subunit. This putative protein kinase A fragment was used to isolate the entire gene from T. cruzi genomic libraries. The deduced 329 amino acid sequence of this gene contained all of the signature motifs of known protein kinase A catalytic subunit proteins. The recombinant protein expressed in Escherichia coli was shown to phosphorylate Kemptide, a synthetic peptide substrate of protein kinase A, in a protein kinase inhibitor (PKI)-inhibitory manner. Immunoprecipitation with polyclonal antisera raised against recombinant protein of this gene was able to pull-down PKI-inhibitory phosphotransferase activity from epimastigote lysates. Immunoblot and Northern blot analyses, in combination with enzyme activity assays, revealed that this gene was a stage-regulated enzyme in T. cruzi with higher levels and activity being present in epimastigotes compared with amastigotes or trypomastigotes. Overall these studies indicate that the cloned gene encodes an authentic protein kinase A catalytic subunit from T. cruzi and are the first demonstration of PKI-inhibitory phosphotransferase activity in an expressed protozoan protein kinase A catalytic subunit.
Huang
Huan
H
Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. huangh@aecom.yu.edu
Werner
Craig
C
Weiss
Louis M
LM
Wittner
Murray
M
Orr
George A
GA
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D017125:Genes, Protozoan / Q000235:genetics; D015151:Immunoblotting; D008969:Molecular Sequence Data; D021122:Protein Subunits; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
D021122:Protein Subunits; D012333:RNA, Messenger; D017868:Cyclic AMP-Dependent Protein Kinases