Phenotypic effects of CD3zeta targeting into glycosphingolipid-enriched membrane microdomains (GEMs) of T cells.
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.
Cebecauer
M
M
Faculty of Sciences, Charles University, Prague, Czech Republic. mcebe@leuko.biomed.cas.cz
Cerný
J
J
D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005786:Gene Expression Regulation; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics; D010641:Phenotype; D019000:Phosphotyrosine / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection; D018909:src Homology Domains / Q000235:genetics
D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D006028:Glycosphingolipids; D007376:Interleukin-2; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)