Our Research Interest
Our primary research interest is in Immunology, with secondary interest in Cancer Biology & Cell Signaling as well as Signal Transduction in lymphocytes. The response of lymphocytes to antigen which initiates an antigen specific immune response also represents a unique opportunity to study how complex molecular interactions between cells can lead to developmental decisions, cell differentiation and proliferation. We are interested in understanding how receptors involved in antigen recognition initiate signal transduction events that regulate cell responses in the immune system.
A sequential model for T-cell activation
Following TCR engagement, CD4-associated Lck is brought into proximity of the CD3 complex and phosphorylates the ITAMs (phoshorylation depicted as red dots). Doubly phosphorylated ITAMs then interact with the tandem SH2 domains of ZAP-70. After ITAM binding, ZAP-70 can be phosphorylated by Lck, which results in activation of ZAP-70 catalytic activity and its autophosphorylation. Active ZAP-70 subsequently phosphorylates LAT and SLP-76, which function as scaffolds to recruit many other signaling molecules and lead to T-cell activation, proliferation, and differentiation (not shown).
SFK activity is reciprocally regulated by Csk and CD45
Phosphorylation of a c-terminal tail negative regulatory tyrosine of SFKs by Csk facilitates its interaction with its SH2 domain, resulting in a closed, catalytically inactive conformation. Dephosphorylation of this site by CD45 favors an open conformation. Phosphorylation of the catalytic site tyrosine is required for full kinase activity. CD45 and PEP can negatively regulate SFK activity by dephosphorylating the catalytic site tyrosine.
Inhibition of ZAP70 activity impairs formation of the immunological synapse
We generated mice that express an engineered mutation of Zap-70 in the catalytic kinase domain. This mutant “analog-sensitive” Zap-70 (or Zap-70(AS)) has kinase activity, but now is also selectively inhibited by a small molecule inhibitor called 3-MB-PP1, an analog of the kinase inhibitor PP1.
In this figure, CD8+ Zap-70(AS) cytotoxic T lymphocytes (CTL) that formed conjugates with target cells were stained for markers of the immunological synapse. We found that CTL were still able to form conjugates if Zap-70 was inhibited with 3-MB-PP1. However, Zap-70 activity is required for clearance of actin from the synapse (inset) and formation of a stable immunological synapse. As a result, inhibition of Zap-70 also severely impairs CTL cytolytic function. Overall these studies show that Zap-70 has catalytic and non-catalytic roles in the formation of immune synapses by CTL.
