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.

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 role for phosphatidylinositol 3-kinase in TCR-stimulated ERK activation leading to paxillin phosphorylation and CTL degranulation.

PI3K is an important regulator of a number of cellular processes. We examined the contribution of PI3K to mouse CTL signaling, leading to degranulation. We show that TCR-triggered, but not phorbol ester and calcium ionophore-induced, CTL degranulation is dependent on PI3K activity. Although PI3K activity is required for optimal LFA-1-mediated adhesion and cell spreading, this most likely does not account for its full contribution to degranulation. We demonstrate that PI3K is required for TCR-stimulated ERK activation in CTL, which we have shown previously to be required for CTL degranulation. We thus define a pathway through which PI3K most likely regulates degranulation and in which ERK appears to be a key signaling molecule. Furthermore, we identified the cytoskeletal adaptor paxillin as a target of ERK downstream of TCR stimulation. Consistent with a role in degranulation, we demonstrate that paxillin is localized to the microtubule organizing center in resting cells and upon target cell binding is recruited to the contact point with the target cell. These studies demonstrate that PI3K regulates ERK activity leading to CTL degranulation, and identify paxillin as a target of ERK downstream of the TCR. That paxillin is independently phosphorylated by both tyrosine kinase(s) and ERK downstream of the TCR and localized both at the microtubule organizing center and at the target cell contact point suggests an important role for paxillin in CTL-mediated killing.