Signal transduction in Entamoeba histolytica induced by interaction with fibronectin: presence and activation of phosphokinase A and its possible relation to invasiveness.
Interaction of Entamoeba histolytica trophozoites with extracellular matrix (ECM) proteins activates signaling pathways through G-protein-coupled receptors. Increments of adenylyl cyclase activity and cAMP produce a striking reorganization of actin into structures that apparently facilitate adhesive, locomotive, and secretory activities. The reorganization of actin is induced by phosphorylation of actin-associated proteins by diverse kinases activated during the signaling process. Although cAMP-dependent kinases have not yet been identified in this parasite, the activation of the adenylyl cyclase route and its effects on particular motility-related functions strongly suggest their presence. Phosphokinase A (PKA) was detected by phosphorylation of the specific substrate, kemptide, its further activation by cAMP, and its inhibition by H89. The catalytic subunit of the enzyme was identified by immunofluorescence microscopy and by immunoprecipitation. Adhesion and damage to cultured cells were monitored by FN-binding and cytotoxicity assays. A cAMP-dependent kinase activated by effectors and agonists of adenylyl cyclase and also during interaction of trophozoites with fibronectin (FN) was found. The enzyme is associated with small granules in the cytoplasm and upon activation, a fraction of its catalytic subunit with an Mr of 100 kDa was translocated to the nucleus, while another fraction was aggregated into big clusters. Activity and translocation were blocked by H89, a specific inhibitor of PKA. Trophozoites stimulated by dBcAMP or forskolin-formed lamellae and restructured actin, but no significant increase in their adhesion to FN was observed and only showed 10% stimulus in their capacity to damage target cells. Treatment with H89 decreased adhesion to 40% and caused 80% inhibition in cell damage. These amebas showed altered organization of the actin structures induced by dBcAMP or FN. Our results support previous suggestions concerning the participation of PKA in the response elicited by the interaction of E. histolytica trophozoites with ECM proteins. They also indicate that adhesion and secretion in conjunction with motile activities are related to invasion processes.
Franco
Elizabeth
E
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados (CINVESTAV) del Instituto Politécnico Nacional, Mexico City, Mexico.
Manning-Cela
Rebeca
R
Meza
Isaura
I
D000199:Actins / Q000378:metabolism; D000818:Animals; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004748:Entamoeba histolytica / Q000166:cytology / Q000378:metabolism* / Q000472:pathogenicity*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D007546:Isoquinolines / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D021122:Protein Subunits; D015398:Signal Transduction / Q000502:physiology*; D013449:Sulfonamides
D000199:Actins; D004791:Enzyme Inhibitors; D005353:Fibronectins; D007546:Isoquinolines; D009842:Oligopeptides; D021122:Protein Subunits; D013449:Sulfonamides; C037642:kemptide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide