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 rec eptor (TCR)-mediated signaling. The absence of ZAP-70 results in impai red differentiation of T cells and a lack of responsiveness to antigen ic stimulation. In order to study the characteristics of ZAP-70 in vit ro, we overexpressed an epitopically tagged human ZAP-70 in a recombin ant baculovirus expression system and purified it by column chromatogr aphy. The kinase activity of purified, recombinant ZAP-70 required cat ion 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 re combinant ZAP-70, unlike that of the homologous protein tyrosine kinas e, Syk, was not affected by binding of TCR-derived tyrosine phosphoryl ated 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 phosphorylati on site, proved to be good substrates, exhibiting K-m values of simila r 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 tow ard enolase, myelin basic protein, calmodulin, histone proteins, or an giotensin could be detected. In contrast to the T cell protein tyrosin e 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 specif icity.