Entamoeba histolytica is a protozoan parasite and the causative agent of amoebiasis in humans. This amoeba invades human tissues by taking advantage of the actin-rich cytoskeleton to move, enter the tissue matrix, kill and phagocyte human cells. During events leading to tissue invasion, E. histolytica moves from the intestinal lumen to cross the mucus layer and enter the epithelial parenchyma. Faced with the chemical and physical constraints of these diverse environments, E. histolytica has developed sophisticated systems to integrate internal and external signals and to coordinate cell shape changes and motility. These cell signalling circuits are driven by the interactions between the parasite and the extracellular matrix combined with rapid responses from the mechanobiome protein network in which protein phosphorylation is important. To understand phosphorylation events we focus on phosphatidylinositol 3-kinases and we have performed live cell imaging and phosphoproteomics. The results highlight 1150 proteins within the amoebic proteome being members of the phosphoproteome, including signalling and structural molecules involved in cytoskeleton activities. Inhibition of phosphatidylinositol 3-kinases alters phosphorylation dynamics in important members of these categories, a finding that correlates with changes in the kinetic parameters of amoeba motility and morphology, as well as with altered content of actin-rich adhesive structures