Rap1b activation and ERK phosphorylation during cAMP/Epac-mediated invasion by Trypanosoma cruzi.

GABRIEL FERRI; DANIEL MUSIKANT; MARTIN M. EDREIRA

Congreso; Molecular Parasitology Meeting XXXI; 2020

Cyclic AMP has been shown to play critical roles during host cell invasion by T. cruzi. Ca2+ release from cellular compartments, such as the endoplasmic reticulum, is accompanied by an elevation of intracellular cAMP levels and it has been shown that cAMP is able to potentiate the Ca2+-dependent exocytosis of lysosomes during host cell invasion. We previously demonstrated that Epac1-mediated signaling represents the main mechanism for cAMP-mediated host cell invasion. Furthermore, Epac1 has been involved in PI3K/Akt and MEK/ERK pathways, and members of these pathways, including Rap1, were localized at late endosomes/lysosomes. In this work, we investigated the involvement of two downstream effectors, Rap1b and ERK, in Epac-mediated invasion. Active GTP-bound Rap1 was detected in lysates from infected cells by pull-down experiments through specific protein interaction with a GST-RalGDS Rap1-binding domain. In line with these results, invasion significantly increased in cells transfected with the constitutively active form of Rap1b (G12V) when compared with the DMSO control and a dominant negative mutant of Rap1b (N17). Regarding ERK participation, we first showed that trypomastigotes induced ERK phosphorylation. In addition, treatment with PD98059, an inhibitor of MEK1/2, suppressed ERK phosphorylation and induced a significant decrease in T. cruzi invasion. Interestingly, co-inhibition of Epac1 and ERK phosphorylation showed no additive or synergistic effects, suggesting that ERK is a downstream effector of cAMP/Epac-mediated invasion.