Comparative phosphorylation of calmodulin from trypanosomatids and bovine brain by calmodulin-binding protein kinases.
Calmodulin (CaM), a major intracellular Ca2+ receptor protein, has been identified and partially characterized in several trypanosomatids. The amino acid sequences of CaM from Trypanosoma cruzi and Trypanosoma brucei are known, while that from Leishmania mexicana is not. CaM from T. cruzi contains 18 amino acid substitutions, as compared with CaM from bovine brain. In addition, CaM from bovine brain contains two tyrosine residues (Tyr-99 and Tyr-138), while CaM from T. cruzi only contains Tyr-138. In the present work we show that a monoclonal antibody developed against the carboxyl-terminal region of bovine brain CaM fails to recognize CaM from both T. cruzi and L. mexicana. CaM from both parasites and from bovine brain were phosphorylated in vitro by a preparation of CaM-binding protein kinases enriched in the epidermal growth factor (EGF) receptor. Phosphoamino acids analysis demonstrated EGF-dependent phosphorylation of tyrosine residues in bovine brain CaM, while only trace amounts of tyrosine phosphorylation were detected in CaM from both trypanosomatids. These results demonstrate that the EGF receptor tyrosine kinase targets Tyr-99, but not Tyr-138, as the single major phosphorylatable residue of CaM. On the other hand, and in contrast to bovine brain CaM, there is a significant phosphorylation of serine residues in CaM from trypanosomatids which is activated by the EGF receptor via a protein-serine/threonine kinase cascade.
Benaim
G
G
Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela. gbenaim@reacciun.ve
Cervino
V
V
Villalobo
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D007894:Leishmania mexicana / Q000201:enzymology*; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D014349:Trypanosoma cruzi / Q000201:enzymology*
D002147:Calmodulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases