EhGEF2, a Dbl-RhoGEF from Entamoeba histolytica has atypical biochemical properties and participates in essential cellular processes.
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.
González De la Rosa
Claudia Haydée
CH
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados, México, DF, Mexico.
Arias-Romero
Luis Enrique
LE
de Jesús Almaraz-Barrera
Ma
M
Hernandez-Rivas
Rosaura
R
Sosa-Peinado
Alejandro
A
Rojo-Domínguez
Arturo
A
Robles-Flores
Martha
M
Vargas
Miguel
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001669:Biochemical Phenomena; D001671:Biochemistry; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D002633:Chemotaxis; D004748:Entamoeba histolytica / Q000235:genetics / Q000378:metabolism*; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D018503:Genome, Protozoan / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010587:Phagocytosis; D010712:Phosphatidic Acids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics; D053058:Trophozoites / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides; D010712:Phosphatidic Acids; D012333:RNA, Messenger; D020741:rho GTP-Binding Proteins