EhFP10: A FYVE family GEF interacts with myosin IB to regulate cytoskeletal dynamics during endocytosis in Entamoeba histolytica.

Motility and phagocytosis are key processes that are involved in invasive amoebiasis disease caused by intestinal parasite Entamoeba histolytica. Previous studies have reported unconventional myosins to play significant role in membrane based motility as well as endocytic processes. EhMyosin IB is the only unconventional myosin present in E. histolytica, is thought to be involved in both of these processes. Here, we report an interaction between the SH3 domain of EhMyosin IB and c-terminal domain of EhFP10, a Rho guanine nucleotide exchange factor. EhFP10 was found to be confined to Entamoeba species only, and to contain a c-terminal domain that binds and bundles actin filaments. EhFP10 was observed to localize in the membrane ruffles, phagocytic and macropinocytic cups of E. histolytica trophozoites. It was also found in early pinosomes but not early phagosomes. A crystal structure of the c-terminal SH3 domain of EhMyosin IB (EhMySH3) in complex with an EhFP10 peptide and co-localization studies established the interaction of EhMySH3 with EhFP10. This interaction was shown to lead to inhibition of actin bundling activity and to thereby regulate actin dynamics during endocytosis. We hypothesize that unique domain architecture of EhFP10 might be compensating the absence of Wasp and related proteins in Entamoeba, which are known partners of myosin SH3 domains in other eukaryotes. Our findings also highlights the role of actin bundling during endocytosis.

Autophosphorylation at Thr279 of Entamoeba histolytica atypical kinase EhAK1 is required for activity and regulation of erythrophagocytosis.

Phagocytosis plays a key role in survival and pathogenicity of Entamoeba histolytica. We have recently demonstrated that an atypical kinase EhAK1 is involved in phagocytosis in this parasite. It is recruited to the phagocytic cups through interaction with EhCaBP1. EhAK1 manipulates actin dynamics by multiple mechanisms including phosphorylation of G-actin. Biochemical analysis showed that EhAK1 is a serine/threonine kinase with broad ion specificity and undergoes multiple trans-autophosphorylation. Three autophosphorylation sites were identified by mass spectrometry. Out of these Thr279 appears to be involved in both autophosphorylation as well as substrate phosphorylation. Over expression of the mutant Thr279A inhibited erythrophagocytosis showing dominant negative phenotype. Multiple alignments of different kinases including alpha kinases displayed conserved binding sites that are thought to be important for function of the protein. Mutation studies demonstrated the importance of some of these binding sites in kinase activity. Binding studies with fluorescent-ATP analogs supported our prediction regarding ATP binding site based on sequence alignment. In conclusion, EhAK1 has multiple regulatory features and enrichment of EhAK1 at the site of phagocytosis stimulates trans-autophosphorylation reaction that increases kinase activity resulting in enhanced actin dynamics and phagocytosis. Some of the properties of EhAK1 are similar to that seen in alpha kinases.