Purification and characterization of human ZAP-70 protein-tyrosine kinase from a baculovirus expression system

The ZAP-70 protein tyrosine kinase is essential for T cell antigen receptor (TCR)-mediated signaling. The absence of ZAP-70 results in impaired differentiation of T cells and a lack of responsiveness to antigenic stimulation. In order to study the characteristics of ZAP-70 in vitro, we overexpressed an epitopically tagged human ZAP-70 in a recombinant baculovirus expression system and purified it by column chromatography. The kinase activity of purified, recombinant ZAP-70 required cation and exhibited a strong preference for Mn2+ over Mg2+. The apparent K-m of ZAP-70 for ATP was similar to 3.0 mu M. The activity of the recombinant ZAP-70, unlike that of the homologous protein tyrosine kinase, Syk, was not affected by binding of TCR-derived tyrosine phosphorylated immunoreceptor tyrosine-based activation motif peptides. Several proteins were tested as potential in vitro substrates of ZAP-70. Only alpha-tubulin and the cytoplasmic fragment of human erythrocyte band 3 (cfb3), which have a region of sequence identity at the phosphorylation site, proved to be good substrates, exhibiting K-m values of similar to 3.3 and similar to 2.5 mu M respectively ([ATP] = 50 mu M). alpha- and beta-Casein were poor substrates for ZAP-70, and no activity toward enolase, myelin basic protein, calmodulin, histone proteins, or angiotensin could be detected. In contrast to the T cell protein tyrosine kinase, Lck, ZAP-70 did not phosphorylate the cytoplasmic portion of the TCR zeta chain or short peptides corresponding to the CD3 epsilon or the TCR zeta immunoreceptor tyrosine-based activation motifs. Our studies suggest that ZAP-70 exhibits a high degree of substrate specificity.