ABSTRACT
Dbl proteins are a family of factors that exchange the guanine nucleotide which promote the activation of Rho small GTPases. This paper reports the molecular, structural, biochemical and functional characterization of EhGEF2, a new member of the Dbl family. EhGEF2 is the second GEF studied in parasites and in the protozoan Entamoeba histolytica, and it is also the first member of the Dbl family that was found to have Arm repeats. The catalytic domain (DH) of EhGEF2 has the conserved residues T421, N590 and E591, which are important for the activation of the GTPases. Biochemical studies on EhGEF2 showed that it could activate in vitro the amoebic GTPases EhRacA, EhRacB, EhRacC, EhRacD, EhRacG, EhRacH and EhCdc42, being EhRacG its main target. It was found that the DH domain binds specifically phosphatidic acid (PA); docking and lipid dot blot studies indicated that this binding does not interfere with the contact surface of EhRacG. Functional studies showed that both the Arm repeats and the catalytic domain of EhGEF2 participate in its localization at the amoebic membrane. Expression of a negative dominant version of EhGEF2 protein in E. histolytica provoked a 30% decrease in its ability to phagocyte human erythrocytes as well as severe effects on both the proliferation and the cellular chemotaxis which suggest that EhGEF2 participates in these cellular processes.
Subject(s)
Entamoeba histolytica/metabolism , Guanine Nucleotide Exchange Factors/metabolism , rho GTP-Binding Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Biochemical Phenomena , Biochemistry , Catalysis , Cell Membrane/metabolism , Cell Proliferation , Cells, Cultured , Chemotaxis , Entamoeba histolytica/genetics , Erythrocytes/cytology , Erythrocytes/metabolism , Gene Expression , Genome, Protozoan/genetics , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotides/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Phagocytosis , Phosphatidic Acids/metabolism , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/genetics , Trophozoites/metabolism , rho GTP-Binding Proteins/geneticsABSTRACT
p21-activated kinases (PAKs) are a highly conserved family of enzymes that are activated by Rho GTPases. All PAKs contain an N-terminal Cdc42/Rac interacting binding (CRIB) domain, which confers binding to these GTPases, and a C-terminal kinase domain. In addition, some PAKs such as Cla4p, Skm1p and Pak2p contain an N-terminal pleckstrin homology (PH) domain and form a distinct group of PAK proteins involved in cell morphology, cell-cycle and gene transcription. Here, we describe a novel p21-activated kinase, denominated EhPAK2, on the parasitic protozoan Entamoeba histolytica. This is the first reported Entamoeba PAK member that contains a N-terminal PH domain and a highly conserved CRIB domain. EhPAK2 CRIB domain shares 29% of amino acid identity and 53% of amino acid homology with these of DdPAKC from Dictyostelium discoideum and Cla4p from Saccharomyces cerevisiae and binds in vitro and in vivo to EhRacA GTPase. This domain also possesses the conserved residues His123, Phe134 and Trp141, which are important for the interaction with the effector loop and strand beta2 of the GTPase; and the residues Met121 and Phe145, which are specific for the interaction of EhPAK2 with EhRacA. Functional studies of EhPAK2 showed that its C-terminal kinase domain had activity toward myelin basic protein. Cellular studies showed that Entamoeba trophozoites transfected with the vector pExEhNeo/kinase-myc, had a 90% decrease in the ability to invade a collagen matrix as well as severe defects in capping, suggesting the involvement of EhPAK2 in these cellular processes.
