The interdependent role of YMNM and PxxP motifs in CD28 costimulation

CD28‐mediated costimualtion is known to be critical required for T cell activation. There are Y189MNM motif and two PxxP motifs in CD28 cytoplasmic region that are thought to play a key role for the costimulatory function. It has been shown that the YMNM motif functions as a binding site for PI3K and Grb2/Gads, and the N‐terminal and C‐terminal PxxP motif play a critical role for binding to Itk and Lck. We generated transgenic mice expressing the Y‐>F mutant CD28 which lack both PI3K and Grb2/Gads binding and P‐>A mutant CD28 which lack binding of Lck and/or Itk in the CD28 deficient background. Previously, we reported that a point mutation of Y189 diminished proliferation and IL‐2 production. In this study, we found that T cells from ncPA mutant mice which have mutations in both N‐ and C‐terminal PxxP motifs showed striking defect in proliferation and IL‐2 production similar to those from Y‐>F mutant. T cells from mice having individual mutation in N‐ or C‐terminal PxxP motif demonstrated a defect of proliferative response to suboptimal dose of stimulation. These results suggested that both N‐ and C‐terminal PxxP motifs are required for CD28 mediated costimulatory functions of T cell activation.