Purification and Characterization of Human ZAP-70 Protein-tyrosine Kinase from a Baculovirus Expression System

doi:10.1074/jbc.271.26.15753

Purification and Characterization of Human ZAP-70 Protein-tyrosine Kinase from a Baculovirus Expression System*

From the ^‡ Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland, 20892, the
^§ Department of Microbiology and Immunology, and the Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84105, Israel, and the
^¶ Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195

^| To whom correspondence should be addressed. Tel.: 301-402-1400; Fax: 301-402-0078; E-mail: samelson{at}helix.nih.gov.

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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TCR

T cell antigen receptor

PTK

protein-tyrosine kinase

ITAM

immunoreceptor tyrosine-based activation motif

SH2

src-homology 2

mAb

monoclonal antibody

kb

kilobase pair(s)

MES

4-morpholineethanesulfonic acid

CAPS

3-(cyclohexylamino)propanesulfonic acid

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

PAGE

polyacrylamide gel electro phoresis

GST

glutathione S-transferase

Ab

antibody

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
↵² J. D. Watts and R. Aebersold, manuscript in preparation.
- Received January 24, 1996.
- Revision received April 15, 1996.