In the present study we tested whether the forced expression of the CD3zeta chain within detergent-resistant, glycosphingolipid-enriched membrane microdomains (GEMs) will result in a constitutively activated phenotype in human T cells. To this aim, a monomeric recombinant protein (LckSH4-CD3zeta), containing the intracellular part of human CD3zeta chain fused to N-terminal double-acylation motif (SH4 domain) of protein tyrosine kinase Lck, was expressed in Jurkat human T lymphoid cell line and its Lck-negative mutant, J. CaM1.6. The Lck SH4 domain indeed predominantly targeted the chimeric protein into GEMs. In transfectants derived from wild-type Jurkat cells, but not in those derived from the Lck-deficient mutant, the LckSH4-CD3zeta protein was constitutively tyrosine-phosphorylated. Tyrosine phosphorylation of a major Jurkat cell phosphoprotein (pp85) was diminished in the transfectants. However, the transfectants did not exhibit any features of constitutively activated T cells, and their responses to anti-CD3 treatment were very similar to the wild-type Jurkat cells. Thus, the constitutive expression of this form of CD3zeta chain in GEMs is not sufficient for eliciting an activated state in the Jurkat cells.

Faculty of Sciences Charles University Prague Czech Republic

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