title: Mannheimia haemolytica leukotoxin activates a nonreceptor tyrosine kinase signaling cascade in bovine leukocytes, which induces biological effects.
abstract: The leukotoxin (LktA) produced by Mannheimia haemolytica binds to bovine lymphocyte function-associated antigen 1 (LFA-1) and induces biological effects in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting any effects. These findings suggest that the specificity of LktA effects must entail both binding to LFA-1 and activation of signaling pathways which are present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA-1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to determine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to examine whether LktA-induced NRTK activation is target cell specific, and (iii) to determine whether LktA-induced NRTK activation is required for biological effects. We used a biologically inactive mutant leukotoxin (DeltaLktA) for comparison with LktA. Our results indicate that LktA induces tyrosine phosphorylation (TP) of the CD18 tail of LFA-1 in bovine leukocytes. The DeltaLktA mutant does not induce TP of the CD18 tail, albeit binding to bovine LFA-1. LktA-induced TP of the CD18 tail was attenuated by an NRTK inhibitor, herbimycin A; a phosphatidylinositol 3'-kinase (PI 3-kinase) inhibitor, wortmannin; and a Src kinase inhibitor, PP2, in a concentration-dependent manner. Furthermore, LktA induces TP of the CD18 tail in bovine, but not porcine, leukocytes. Moreover, LktA-induced intracellular calcium ([Ca2+]i) elevation was also inhibited by herbimycin A, wortmannin, and PP2. Thus, our data represent the first evidence that binding of LktA to bovine LFA-1 induces a species-specific NRTK signaling cascade involving PI 3-kinase and Src kinases and that this signaling cascade is required for LktA-induced biological effects.
authors.affiliation: Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108, USA.
authors.forename: S
authors.initials: S
authors.lastname: Jeyaseelan
authors.forename: M S
authors.initials: MS
authors.lastname: Kannan
authors.forename: R E
authors.initials: RE
authors.lastname: Briggs
authors.forename: P
authors.initials: P
authors.lastname: Thumbikat
authors.forename: S K
authors.initials: SK
authors.lastname: Maheswaran
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D016227:Benzoquinones; D018821:CD18 Antigens / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D047029:Lactams, Macrocyclic; D007962:Leukocytes / Q000166:cytology / Q000378:metabolism; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000378:metabolism; D016978:Mannheimia haemolytica / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D016227:Benzoquinones; D018821:CD18 Antigens; D005098:Exotoxins; D006460:Hemolysin Proteins; D047029:Lactams, Macrocyclic; D016169:Lymphocyte Function-Associated Antigen-1; D011809:Quinones; C102677:lktA protein, bacteria; D017828:Rifabutin; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D002118:Calcium; D000077191:Wortmannin