EhPAK2, a novel p21-activated kinase, is required for collagen invasion and capping in Entamoeba histolytica.
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.
Arias-Romero
Luis Enrique
LE
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios, Avanzados del IPN, Mexico, DF, Mexico.
de Jesús Almáraz-Barrera
Ma
M
Díaz-Valencia
Juan Daniel
JD
Rojo-Domínguez
Arturo
A
Hernandez-Rivas
Rosaura
R
Vargas
Miguel
M
D000595:Amino Acid Sequence; D000818:Animals; D003094:Collagen / Q000378:metabolism*; D048749:Cytokinesis; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D004789:Enzyme Activation; D007152:Immunologic Capping; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000378:metabolism; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D014162:Transfection; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism
D004676:Myelin Basic Protein; D003094:Collagen; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins