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.

A signaling network stimulated by β 2 integrin promotes the polarization of lytic granules in cytotoxic cells

“…Paxillin is a cytoskeletal scaffold protein with multiple interaction domains, which contributes to β 2 integrin-dependent granule polarization in NK cells (5). In T cells, paxillin localizes to the peripheral region of the immunological synapse, binds to microtubules, and contributes to MTOC reorientation (32). Therefore, paxillin may also provide a connection with the microtubular network in NK cells stimulated by β 2 integrin.…”

Controlling Natural Killer Cell Responses: Integration of Signals for Activation and Inhibition

“…Pyk2 is required for the reorientation of the MTOC and paxillin in NK cells that are in contact with sensitive target cells (121). Studies of T cell immunological synapses have shown that paxillin is bound to the MTOC and that the combined signals from LFA-1 and TCR result in paxillin recruitment to the site of integrin engagement (122). This is consistent with the role of paxillin in the LFA-1--dependent polarization of granules in NK cells (123).…”

Pyk2 Controls Integrin-Dependent CTL Migration through Regulation of De-Adhesion

“…CTL nucleofection was performed, as described previously (28), with the modification that at day 4 post-CTL stimulation, live cells were enriched with Histopaque-1077 (Sigma-Aldrich, Mississauga, Ontario, Canada) prior to nucleofection. Approximately 1.5 3 10 6 million cells were used for each nucleofection, as per the specifications described by the manufacturer for the mouse T cell kit (Lonza, Basel, Switzerland).…”