Syk Mutation in Jurkat E6-derived Clones Results in Lack of p72syk Expression

Joseph Fargnoli,Anne L Burkhardt,Maureen Laverty,Stephanie A Kut,Nicolai S.C Van Oers,Arthur Weiss,Joseph B Bolen

doi:10.1074/jbc.270.44.26533

Abstract

The human leukemic Jurkat cell line is commonly used as a model cellular system to study T lymphocyte signal transduction. Various clonal derivatives of Jurkat T cells exist which display different characteristics with regard to responses to external stimuli. Among these, the E6-1 clone of Jurkat T cells has been used as a parental line from which numerous important somatic mutant clones have been generated. During the course of experiments examining signals initiated by the T cell antigen receptor in an E6-1-derived Jurkat cell clone J.CaM1, we observed that the 72-kilodalton Syk protein tyrosine kinase previously found in other Jurkat cells was not detected. Upon further analysis it was determined that Syk transcripts from the J.CaM1 cells as well as the parental E6-1 cells contain a single guanine nucleotide insertion at position 92. This nucleotide insertion results in a shift in the Syk open reading frame leading to alternate codon usage as well as the generation of a termination codon at position 109. Thus, Syk transcripts in E6-1 cells and E6-1-derived clones are predicted to be capable of encoding only the first 33 amino acids of the 630-amino acid wild type Syk. These findings are incompatible with a recently proposed model of T cell antigen receptor signal transduction based, in part, on experiments conducted using E6-1-derived cells, suggesting that Syk might play a role upstream of Lck and Zap70.

Highlights

The Syk protein tyrosine kinase together with the Zap70 protein tyrosine kinase comprise a family of cytoplasmic enzymes that are important for signal transduction initiated by different types of surface receptors in cells of hemopoietic origin [1]
During the course of experiments examining protein tyrosine kinase signaling in the J.CaM1 somatic cell mutant isolated from the Jurkat E6-1 line [24], we noticed that the 72-kDa Syk protein tyrosine kinase was not readily detected
Lack of Detectable p72syk in E6-1-derived Jurkat Cells—Representative Jurkat lines were examined for the expression of Zap70 and Syk by specific enzyme immunoprecipitation followed by immunoblot analysis

Summary

Introduction

The Syk protein tyrosine kinase together with the Zap protein tyrosine kinase comprise a family of cytoplasmic enzymes that are important for signal transduction initiated by different types of surface receptors in cells of hemopoietic origin [1]. The Syk and Zap SH2 domains serve to bind tandem phosphotyrosine containing elements in the membrane-associated signal coupling subunits of immune recognition receptors [1]. These 18 –20 amino acid elements are referred to as immunoreceptor tyrosine activation motifs and have the consensus sequence (D/E)XXYXX(I/L)X6–8YXX(I/L) [4]. Patients with mutations in the ZAP gene demonstrate abnormal development of thymocytes leading to the production of exclusively CD4ϩ T cells in the periphery (19 –21). These T cells are, unresponsive to mitogen and antigen stimulation (19 –21). These results demonstrate that functional Syk is not expressed in all Jurkat T cell-derived clones

Methods

Results

Conclusion