Regulation of Ca2+/calmodulin-dependent protein kinase from Trypanosoma cruzi.
A multifunctional Ca2+/calmodulin-dependent protein kinase (TcCaM K) was purified and characterized from the cytosol of Trypanosoma cruzi epimastigote forms. Like mammalian CaM KII, TcCaM K has a broad substrate specificity and a similar subunit composition. Western blot analysis revealed that this TcCaM K possesses two subunits of 50 and 60 kDa, which exhibited autophosphorylating activity. A panel of monoclonal and polyclonal antibodies raised against rat brain CaM KII could also recognize TcCaM K. However, experimental evidence suggests a different conformational arrangement of the TcCaM K subunits. Like its mammalian counterpart, two highly active autonomous, Ca(2+)-independent, states of TcCaM K can be isolated. These states, caused by high phosphate incorporation, differ only in their extent of Ca2+/CaM-dependence. About 15-20% of the autophosphorylated TcCaM K can be reverted using protein phosphatase 2A, and, consequently, its Ca(2+)-dependent activity is also partially restored. The situation is somewhat different when the enzyme is linked to the cytoskeleton, as we have previously shown. The membrane-bound form is present only in the native form. Activation increases its protein kinase activity from 5- to 14-fold. In this study, we provide evidence of another form of TcCaM K present in soluble fractions of the parasite that can be isolated in autonomous states. Our results suggest that autophosphorylation of membrane-bound TcCaM K may be responsible for kinase release in a Ca2+/CaM-independent state. These properties of TcCaM K may play an important role in regulating Ca(2+)-dependent processes in the parasite.
Ogueta
S
S
Instituto de Investigaciones en Ingeniería Genética y Biología, Molecular (INGEBI, CONICET), Buenos Aires, Argentina.
Intosh
G M
GM
Téllez-Iñon
M T
MT
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D051381:Rats; D012995:Solubility; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C063391:syntide-2; D017871:Calcium-Calmodulin-Dependent Protein Kinases