Benaim G, Cervino V, Villalobo A
Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela.
Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1998 Jul;120(1):57-65. doi: 10.1016/s0742-8413(98)00006-1.
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.
钙调蛋白（CaM）是一种主要的细胞内钙离子受体蛋白，已在几种锥虫中得到鉴定并进行了部分特性分析。克氏锥虫和布氏锥虫的钙调蛋白氨基酸序列已知，而墨西哥利什曼原虫的钙调蛋白氨基酸序列未知。与牛脑钙调蛋白相比，克氏锥虫的钙调蛋白含有18个氨基酸替换。此外，牛脑钙调蛋白含有两个酪氨酸残基（Tyr-99和Tyr-138），而克氏锥虫的钙调蛋白仅含有Tyr-138。在本研究中，我们发现一种针对牛脑钙调蛋白羧基末端区域制备的单克隆抗体无法识别克氏锥虫和墨西哥利什曼原虫的钙调蛋白。来自这两种寄生虫以及牛脑的钙调蛋白在体外被富含表皮生长因子（EGF）受体的钙调蛋白结合蛋白激酶制剂磷酸化。磷酸氨基酸分析表明，牛脑钙调蛋白中的酪氨酸残基发生了EGF依赖性磷酸化，而在两种锥虫的钙调蛋白中仅检测到微量的酪氨酸磷酸化。这些结果表明，EGF受体酪氨酸激酶将Tyr-99而非Tyr-138作为钙调蛋白唯一主要的可磷酸化残基。另一方面，与牛脑钙调蛋白不同，锥虫钙调蛋白中的丝氨酸残基存在显著磷酸化，这是由EGF受体通过蛋白丝氨酸/苏氨酸激酶级联反应激活的。
