Proline residues in CD28 and the Src homology (SH)3 domain of Lck are required for T cell costimulation

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation.