title: Paxillin associates with the microtubule cytoskeleton and the immunological synapse of CTL through its leucine-aspartic acid domains and contributes to microtubule organizing center reorientation.
abstract: The cytoskeletal adaptor protein paxillin localizes to the microtubule organizing center (MTOC) in T cells and, upon target cell binding, is recruited to the supramolecular activation complex (SMAC). We mapped the region of paxillin that associates with both the MTOC and SMAC to the leucine-aspartic acid (LD) domains and showed that a protein segment containing LD2-4 was sufficient for MTOC and SMAC recruitment. Examination of the localization of paxillin at the SMAC revealed that paxillin localizes to the peripheral area of the SMAC along with LFA-1, suggesting that LFA-1 may contribute to its recruitment. LFA-1 or CD3 engagement alone was insufficient for paxillin recruitment because there was no paxillin accumulation at the site of CTL contact with anti-LFA-1- or anti-CD3-coated beads. In contrast, paxillin accumulation was detected when beads coated with both anti-CD3 and anti-LFA-1 were bound to CTL, suggesting that signals from both the TCR and LFA-1 are required for paxillin accumulation. Paxillin was shown to be phosphorylated downstream of ERK, but when we generated a mutation (S83A/S130A) that abolished the mobility shift as a result of phosphorylation, we found that paxillin still bound to the MTOC and was recruited to the SMAC. Furthermore, ERK was not absolutely required for MTOC reorientation in CTL that require ERK for killing. Finally, expression of the LD2-4 region of paxillin substantially reduced MTOC reorientation. These studies demonstrated that paxillin is recruited, through its LD domains, to sites of integrin engagement and may contribute to MTOC reorientation required for directional degranulation.
authors: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton T6G 2S2, Alberta, Canada.
authors: Leslie K
authors: LK
authors: Robertson
authors: Hanne L
authors: HL
authors: Ostergaard
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D015550:Cell Degranulation / Q000502:physiology; D054992:Immunological Synapses / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D047468:Immunoprecipitation; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D008213:Lymphocyte Activation / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D022101:Microtubule-Organizing Center / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D051419:Paxillin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D051419:Paxillin; D001224:Aspartic Acid; D007930:Leucine