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 Km of ZAP-70 for ATP was approximately 3.0 microM. 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 Kmvalues of approximately 3.3 and approximately 2.5 microM, respectively ([ATP] = 50 microM). 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 TCRzeta chain or short peptides corresponding to the CD3epsilon or the TCRzeta immunoreceptor tyrosine-based activation motifs. Our studies suggest that ZAP-70 exhibits a high degree of substrate specificity.
Isakov
N
N
Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland, 20892, USA.
Wange
R L
RL
Watts
J D
JD
Aebersold
R
R
Samelson
L E
LE
D000595:Amino Acid Sequence; D016367:Baculoviridae; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000302:isolation & purification*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D008345:Manganese; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D008274:Magnesium