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

Noah Isakov, Ronald L. Wange, Julian D. Watts, Ruedi Aebersold, Lawrence E. Samelson

Research output: Contribution to journalArticlepeer-review

54 Scopus citations

Abstract

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 ~3.0 μM. 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 α-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 ~3.3 and ~2.5 μM, respectively ([ATP] = 50 μM). α- and β-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 TCRζ chain or short peptides corresponding to the CD3ε or the TCRζ immunoreceptor tyrosine-based activation motifs. Our studies suggest that ZAP-70 exhibits a high degree of substrate specificity.

Original languageEnglish
Pages (from-to)15753-15761
Number of pages9
JournalJournal of Biological Chemistry
Volume271
Issue number26
DOIs
StatePublished - 16 Jul 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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