CD4:p56lck association studied in vivo using antibody-induced capping and double indirect immunofluorescence microscopy.
Accumulating data suggest that the T-cell surface antigen CD4 transduces an independent signal during antigen-mediated T-cell activation. In vitro studies which showed that the cytoplasmic protein tyrosine kinase p56lck is present in anti-CD4 immunoprecipitates led to the model that p56lck is associated with the cytoplasmic domain of CD4. In this report we have extended these studies and examined potential CD4:p56lck associations in vivo. We show here by double immunofluorescence microscopy a specific co-distribution of p56lck with antibody-induced CD4 caps in intact cells. Murine T-cell hybridoma lines expressing mutant forms of CD4 were used to demonstrate that the 31 carboxyterminal aminoacids of its cytoplasmic domain, in particular cysteine-420 and cysteine-422, are crucial for the formation of CD4:p56lck complexes in vivo. The potential of the method applied is discussed with regard to studies of other transmembrane signalling systems involving src-like kinases.
Gassmann
M
M
Department of Biology, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Amrein
K E
KE
Burn
P
P
D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry*; D002470:Cell Survival / Q000276:immunology; D003545:Cysteine / Q000032:analysis; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007152:Immunologic Capping / Q000502:physiology*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011505:Protein-Tyrosine Kinases / Q000032:analysis*
D015704:CD4 Antigens; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D003545:Cysteine