Characterization of tyrosine phosphorylation in the protein tyrosine phosphatase CD45

The enzymatic activity of the protein tyrosine phosphatase, CD45 has been demonstrated to play an absolutely required role in the regulation of Src family protein tyrosine kinases in T lymphocytes. CD45 function during early events of antigen receptor signaling has been well established, however, the role of CD45 during later stages of T cell activation is only beginning to emerge. Transient tyrosine phosphorylation of CD45 has previously been demonstrated. We show this phosphorylation can be sustained through treatment of a T cell hybridoma cell line with the PTPase inhibitor, pervanadate. Tyrosine phosphorylation of CD45 is significantly increased in the presence of an activated form of Lck. Tyrosine phosphorylated CD45 is correlated with the association of a subset of signaling molecules containing SH2 domains. These molecules include p56Lck, p59Fyn, rasGAP, Grb2 and Csk. Although CD45 becomes abundantly tyrosine phosphorylated upon pervanadate treatment, no other SH2-containing molecules that we had tested demonstrated association with CD45. The interaction between CD45 and Grb2 in particular was found to be dependent upon CD45 tyrosine phosphorylation and mediated through the SH2 domain of Grb2. Interestingly, both Grb2 and a close homolog, Grap are able to bind CD45 in in vitro binding assays. Grap does not, however, associate with CD45 in vivo as was observed for Grb2.CD45 contains two Grb2 consensus binding sequences. Deletion of both results in a greatly diminished capacity for CD45- Grb2 association. However, interaction between the two is not completely abolished and appears to result from an unconventional peptide sequence as observed by 2-dimensional phosphopeptide mapping studies. In the process of mapping the phosphotyrosine sequences required for CD45-Grb2 interaction, many tyrosine-phosphorylated peptides have been identified and assigned to tyrosine residues in CD45