Molecular and biochemical characterization of a protein kinase B from Trypanosoma cruzi.
A Trypanosoma cruzi gene, PKB, coding for a putative protein kinase was cloned and sequenced. Analysis of the sequence showed that the encoded protein (called PKB) corresponds to a relatively novel subgroup of Ser/Thr protein kinases denominated protein kinases B (PKB), related to A and C protein kinases (RAC), or protein kinases of the transforming retrovirus AKT8 (Akt) in which the catalytic domains show similarity to corresponding domains of protein kinases A and protein kinases C. Unlike mammalian enzymes belonging to the same subgroup, PKB did not have a pleckstrin (PH)-homologous domain. PKB was expressed in Escherichia coli and the recombinant protein was found to be a Thr-specific protein kinase that required Mn2+ for activity and used ATP as phosphate donor (Km = 1.8 microM). Classical protein kinase A and protein kinase C modulators and inhibitors were found to have only marginal or no effect on PKB activity. Antisera raised against the recombinant protein recognized PKB in Western blotting analysis of cell extracts as a membrane bound protein. Evidence was obtained suggesting the presence of a Cys-linked acyl anchor. Northern and Western blotting analysis showed that PKB was constitutively expressed in the lag, exponential and stationary phases of T. cruzi epimastigote growth, as well as in the amastigote and metacyclic trypomastigote stages of differentiation. This is the first description of the existence of a protein kinase B in trypanosomatid protozoa.
Pascuccelli
V
V
Instituto de Investigaciones Bioquímicas Fundación Campomar, Buenos Aires, Argentina.
Labriola
C
C
Téllez-Iñón
M T
MT
Parodi
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D016054:DNA, Protozoan; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000379:methods; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D016054:DNA, Protozoan; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt