guanosine-5--o-(3-thiotriphosphate) and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Philadelphia chromosome-positive leukemias result from the fusion of the BCR and ABL genes, which generates a functional chimeric molecule. The Abr protein is very similar to Bcr but lacks a structural domain which may influence its biological regulatory capabilities. Both Abr and Bcr have a GTPase-activating protein (GAP) domain similar to those found in other proteins that stimulate GTP hydrolysis by members of the Rho family of GTP-binding proteins, as well as a region of homology with the guanine nucleotide dissociation-stimulating domain of the DBL oncogene product. We purified as recombinant fusion proteins the GAP- and Dbl-homology domains of both Abr and Bcr. The Dbl-homology domains of Bcr and Abr were active in stimulating GTP binding to CDC42Hs, RhoA, Rac1, and Rac2 (rank order, CDC42Hs > RhoA > Rac1 = Rac2) but were inactive toward Rap1A and Ha-Ras. Both Bcr and Abr acted as GAPs for Rac1, Rac2, and CDC42Hs but were inactive toward RhoA, Rap1A, and Ha-Ras. Each individual domain bound in a noncompetitive manner to GTP-binding protein substrates. These data suggest the multifunctional Bcr and Abr proteins might interact simultaneously and/or sequentially with members of the Rho family to regulate and coordinate cellular signaling.

Topics: Binding, Competitive; Cloning, Molecular; Escherichia coli; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutagenesis, Insertional; Oncogene Proteins; Oncogenes; Protein Biosynthesis; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcr; rac GTP-Binding Proteins; Recombinant Fusion Proteins

In the present work, anti-G protein antibodies were introduced inside streptolysin permeabilized O interleukin-2-activated natural killer (IANK) cells. Successful entry of the antibodies was determined by flow cytometry and fluorescence microscopy. Permeabilized cells showed typical large granular lymphocyte morphology and remained functional, significantly lysing both NK-sensitive K562 cells and NK-resistant/IANK-sensitive RAJI target cells. This method was utilized to study the effect of anti-G protein antibodies on the functional activities of IANK cells. Anti-Gs antibody inhibited IANK cell killing of RAJI but not of K562 target cells. Further analysis showed that K562 and RAJI cells enhance the binding of guanosine 5'-O-(thiotriphosphate) to IANK cell membranes, and increase the hydrolysis of [32P]GTP in these membranes. Immunoblot analysis showed that K562 and RAJI cells induce the release of alpha o, but not alpha i, alpha s, or alpha q.11 from IANK cell membranes. Cumulatively, these data indicate that putative receptors recognizing K562 or RAJI target cells are coupled to Go in IANK cells, however, only Gs seems to be coupled to receptors recognizing RAJI target cells. Our results point out the importance of Gs protein as a mediator of cellular cytotoxicity of the anti-tumor effector cells.

Topics: Animals; Antibodies; Bacterial Proteins; Burkitt Lymphoma; Cell Line; Cell Membrane Permeability; Cytotoxicity, Immunologic; Flow Cytometry; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Immunoglobulin G; Interleukin-2; Killer Cells, Natural; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphocyte Activation; Rabbits; Streptolysins; Tumor Cells, Cultured