Characterization of Vps15: a putative regulatory kinase of Vps34 in Trypanosoma cruzi.

SCHOIJET AC; FLAWIÁ MM; ALONSO GD

Potrero de los Funes, San Luis.

Congreso; XLVII Reunión Anual de SAIB; 2011

SAIB

Class III phosphoinositide 3-kinases (PI3Ks), known as Vps34, are important regulators of vesicular trafficking, autophagy and nutrient sensing. Previously, we have functionally characterized the first class III PI3K reported in Trypanosoma cruzi, named TcVps34, which has a role in vital processes, such as osmoregulation, acidification, and receptor-mediated endocytosis. In yeast and other organisms, Vps34 exerts its lipid kinase activity by forming a protein complex with a Ser/Thr protein kinase known as Vps15. In this work, we initiated the characterization of TcVps15, its homologue in T. cruzi. The recombinant TcVps15 was able to phosphorylate both the comertial histone mix (H2As) and the myelin basic protein, showing a cation preference for Mn+2. Moreover, transgenic parasites overexpressing TcVps15 showed an increase in their kinase activity comparing to wild type cells. In addition, western blot analysis of T. cruzi parasites overexpressing TcVps34 or TcVps15 showed that both enzymes are localized mainly at the membrane fraction. However, both proteins were partially released with high salt concentration. Interestingly, the localization of these proteins was not affected by the treatment with wortmannin, a PI3K inhibitor. Further experiments will be needed to address the mechanism by which TcVps15 regulates TcVps34, as well as the function of the kinase activity of TcVps15.