Efficient T-cell activation requires two signals, one emanating from the antigen-specific T-cell receptor, and the other from the CD28 cell surface receptor. The net result of these two signals is the up-regulation of cytokines and other molecules required for efficient T-cell expansion. While analysis of CD28 signals has revealed roles for Lck (a member of the Src family on non-receptor tyrosine kinases) and ITK (a member of the Tec family of protein tyrosine kinases), there remain significant gaps in knowledge on how CD28 activates these two kinases. The overall aim of the present project is to determine the molecular interactions responsible for activation of ITK by CDC28. The hypothesis is that CD28 regulates ITK by the concerted action of Src family kinases and the control over the recruitment and binding of ITK to CD28. This hypothesis will be tested by pursuing two specific aims: (1) to identify the domains of ITK required for its efficient activation by CD28, and conversely (2) to determine the specific residues within CD28 required for activation of ITK. The approach will be to express deletion and other mutational constructs of the two proteins in a B-cell line that lacks endogenous CD28 and Tec kinases. The information to be gained will significantly expand the knowledge of the regulatory mechanisms of the Tec family of protein kinases.
