Rho family GTPases modulate actin cytoskeleton dynamics by signaling through multiple effectors including the p21-activated kinases (PAKs). A structural assessment with EhRho1 in complex with EhFormin1 suggests likely determinants of Rho family GTPase signaling specificity in is the causative agent of amoebic colitis and systemic amoebiasis.14 Encysted is spread primarily through GX15-070 contaminated food and water sources among poor populations of developing countries although outbreaks among travelers and susceptible populations occur in the United States.14cysts cycle to the trophozoite form in the human being intestine and may give rise to local destruction of the intestinal mucosa (amoebic colitis) or more rarely may enter the bloodstream leading to systemic amoebiasis characterized by liver lung and mind abscesses.15 The pathogenesis of infection depends on a highly dynamic actin-rich trophozoite cytoskeleton.16 Single-cell trophozoites communicate ～20 Rho family GTPases and downstream signaling effectors important for coordination of actin cytoskeletal rearrangement in pathogenesis-related processes including migration and chemotaxis adherence to intestinal epithelium and sponsor cell killing and phagocytosis (examined in ref (17)). For instance manifestation of constitutively active EhRacA or EhRacG in trophozoites alters phagocytosis and surface receptor capping 18 19 while EhRho1 engages a diaphanous-related formin effector EhFormin1 to directly modulate actin polymerization.20 21 EhRacC directly interacts with the heterotrimeric G protein effector EhRGS-RhoGEF and together with EhGα1 promotes Rac GTPase activation in cells.22 Six PBD-containing kinases related to mammalian PAKs will also be encoded from the genome.17 23 An additional protein EhPAK (also called EhPAK1) does not contain a conserved PBD but was found to bind human being Rac1 at its N-terminus.24 EhPAK1 localizes to the leading edge of migrating trophozoites and is implicated in amoeboid migration polarity and human being red blood cell phagocytosis.24 EhPAK2 has a part in collagen matrix invasion and its PBD selectively engages activated EhRacA.17 23 A third analyzed PAK EhPAK3 autophosphorylates and displays kinase activity in the absence of small GTPases. 25 Therefore PAKs regulate pathogenesis-related processes particularly trophozoite migration and extracellular matrix invasion. However the relationship of PAK isoforms to mammalian PAKs remains unclear; specifically it is not known how their activation mechanisms are related to mammalian group I and group II modes of autoinhibition. The degree of Rho family GTPase/PAK signaling specificity in is also an unresolved query given the apparent simultaneous manifestation of ～20 Rho family GTPases and up to seven PAKs inside a single-cell organism. Here we quantify the GTPase binding selectivity of two previously unstudied PAKs and determine the structural relationship of the EhRacC/EhPAK4 PBD interface to mammalian homologues. Experimental Methods Cloning and Protein Purification Genomic DNA was isolated from your virulent HM-1:IMSS strain of using a DNeasy Blood and Tissue Kit (Qiagen). EhRho1 EhRacC EhRacD and EhRacG were cloned from genomic DNA by polymerase chain reaction (PCR) amplification as hexahistidine-tagged Rabbit Polyclonal to CYSLTR2. open reading framework fusions indicated in B834 prenylation motif (11 residues) was excluded and a glutamine (Q65) required for GTPase activity was mutated to leucine using the two-PCR method.26 The EhRacCQ65L N-terminal hexahistidine tag was removed with tobacco etch virus (TEV) prior to NTA affinity chromatography and gel filtration as described previously for EhRho1.20 Open reading frames of the isolated p21 binding domains (PBDs) of EhPAK4 (EHI_152540 amino acids 12 and EhPAK5 (EHI_043140 amino acids 105-161) were amplified via PCR from genomic DNA and subcloned as hexahistidine fusions into GX15-070 a pET vector-based ligation-independent cloning vector pLIC-His as explained previously.20 The following PCR primer sequences GX15-070 were used: EhPAK4 5 and 5′-TTATGTTCTATTTCCATTATC-3′; and EhPAK5 5 GX15-070 and 5′-TTATTGTGTGAATTCTAATAC-3′. For each PAK B834 cells were grown to an OD600 of 0.8 at 37 °C and expression was induced with 500 mM isopropyl β-d-thiogalactopyranoside (IPTG) for 14-16 h at 20 °C. Pelleted bacterial cells were resuspended in N1 buffer comprising 30 mM HEPES (pH 8.0) 250 mM NaCl and 30 mM imidazole and lysed by high-pressure homogenization with an Emulsiflex (Avestin Ottawa ON). Cellular lysates were cleared by centrifugation at 100000for 1 h at 4 °C and the.